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Abstract:

Disclosed are methods for detecting non-small cell lung cancer (NSCLC)
using differentially expressed genes KIF11, GHSR1b, NTSR1, and FOXM1.
Also disclosed are methods of identifying compounds for treating and
preventing NSCLC, based on the interaction between KOC1 and KIF11, or NMU
and GHSR1b or NTSR1.

Claims:

1. A method of screening for a compound for treating or preventing NSCLC,
said method comprising the steps of: contacting a KIF11 polypeptide or
functional equivalent thereof with KOC1 polypeptide or functional
equivalent thereof in the presence of a test compound; detecting the
binding between the polypeptides; and selecting the test compound that
inhibits the binding between the polypeptides.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Ser. No.
12/700,669, filed Feb. 4, 2010, which claims priority to U.S. Ser. No.
10/593,842, filed Jul. 10, 2007, now U.S. Pat. No. 7,700,573, which is a
U.S. National Phase of International Application No. PCT/JP2005/005613,
filed Mar. 18, 2005, which claims priority to U.S. Ser. No. 60/555,789
filed Mar. 23, 2004, all of which are incorporated herein by reference in
their entireties.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of biological science,
more specifically to the field of cancer therapy and diagnosis. In
particular, the invention relates to methods of diagnosing non-small cell
lung cancers using genes, KIF11, GHSR1b, NTSR1, and FOXM1, that show
elevated expression in such cancerous cells.

BACKGROUND OF THE INVENTION

[0003] Lung cancer is one of the most commonly fatal human tumors. Many
genetic alterations associated with the development and progression of
lung cancer have been reported. Although genetic changes can aid
prognostic efforts and predictions of metastatic risk or response to
certain treatments, information about a single or a limited number of
molecular markers generally fails to provide satisfactory results for
clinical diagnosis of non-small cell lung cancer (NSCLC) (Mitsudomi et
al., Clin Cancer Res 6: 4055-63 (2000); Niklinski et al., Lung Cancer. 34
Suppl 2: S53-8 (2001); Watine, BMJ 320: 379-80 (2000)). NSCLC is by far
the most common form, accounting for nearly 80% of lung tumors (Society,
A.C. Cancer Facts and Figures 2001 (2001)). The overall 10-year survival
rate remains as low as 10% despite recent advances in multi-modality
therapy, because the majority of NSCLCs are not diagnosed until advanced
stages (Fry, W. A. et al., Cancer 86: 1867-76 (1999)). Although
chemotherapy regimens based on platinum are considered the reference
standards for treatment of NSCLC, those drugs are able to extend survival
of patients with advanced NSCLC only about six weeks (Non-small Cell Lung
Cancer Collaborative Group, BMJ. 311: 899-909 (1995)). Numerous targeted
therapies are being investigated for this disease, including tyrosine
kinase inhibitors, but so far promising results have been achieved in
only a limited number of patients and some recipients suffer severe
adverse reactions (Kris M. N. R., Herbst R. S. Proc. Am. Soc. Clin.
Oncol. 21: 292a(A1166) (2002)).

[0004] Many genetic alterations associated with development and
progression of lung cancer have been reported, but the precise molecular
mechanisms remain unclear (Sozzi, G. Eur. J. Cancer 37: 63-73 (2001)).
Over the last decade newly developed cytotoxic agents including
paclitaxel, docetaxel, gemcitabine, and vinorelbine have emerged to offer
multiple therapeutic choices for patients with advanced NSCLC; however,
each of the new regimens can provide only modest survival benefits
compared with cisplatin-based therapies (Schiller, J. H. et al., N. Engl.
J. Med. 346: 92-98 (2002); Kelly, K. et al., J. Clin. Oncol. 19:
3210-3218 (2001)). Hence, new therapeutic strategies, such as development
of molecular-targeted agents, are eagerly awaited by clinicians.

[0008] KOC1 is orthologous to the Xenopus Vg1 RNA-binding protein
(Vg1RBP/Vera), which mediates the localization of Vg1 mRNA to the vegetal
pole of the oocyte during oocyte maturation, and IMP-1 is orthologous to
the ZBP1. IMP is mainly located at the cytoplasm and its cellular
distribution ranges from a distinct concentration in perinuclear regions
and lamellipodia to a completely delocalized pattern. H19 RNA
co-localized with IMP, and removal of the high-affinity attachment site
led to delocalization of the truncated RNA (Runge, S. et al., J. Biol.
Chem. 275: 29562-29569 (2000)), suggesting that IMPs are involved in
cytoplasmic trafficking of RNA. IMP-1 was able to associate with
microtubles (Nielsen, F. C. et al., J. Cell Sci. 115: 2087-2097 (2002);
Havin, L. et al., Genes Dev. 12: 1593-1598 (1998)), and is likely to
involve a motor protein such as kinesin, myosin, and dyenin. On the other
hand, Oskar mRNA localization to the posterior pole requires Kinesin I
(Palacios, I. M., St. Johnston D. Development 129: 5473-5485 (2002);
Brendza, R. P. et al., Science 289: 2120-2102 (2000)).

[0009] KIF11 (alias EG5) is a member of kinesin family, and plays a role
in establishing and/or determining the stability of specific microtuble
arrays that form during cell division. This role may encompass the
ability of KIF11 to influence the distribution of other protein
components associated with cell division (Whitehead, C. M., Rattner, J.
B. J. Cell Sci. 111: 2551-2561 (1998); Mayer, T. U. et al., Science 286:
971-974 (1999)).

[0015] It has been repeatedly reported that peptide-stimulated peripheral
blood mononuclear cells (PBMCs) from certain healthy donors produce
significant levels of IFN-γ in response to the peptide, but rarely
exert cytotoxicity against tumor cells in an HLA-A24 or -A0201 restricted
manner in 51Cr-release assays (Kawano et al., Cancer Res. 60: 3550-8
(2000); Nishizaka et al., Cancer Res. 60: 4830-7 (2000); Tamura et al.,
Jpn. J. Cancer Res. 92: 762-7 (2001)). However, both of HLA-A24 and
HLA-A0201 are one of the popular HLA alleles in Japanese, as well as
Caucasian (Date et al., Tissue Antigens 47: 93-101 (1996); Kondo et al.,
J. Immunol. 155: 4307-12 (1995); Kubo et al., J. Immunol. 152: 3913-24
(1994); Imanishi et al., Proceeding of the eleventh International
Histocompatibility Workshop and Conference Oxford University Press,
Oxford, 1065 (1992); Williams et al., Tissue Antigen 49: 129 (1997)).
Thus, antigenic peptides of cancers presented by these HLAs may be
especially useful for the treatment of cancers among Japanese and
Caucasian. Further, it is known that the induction of low-affinity CTL in
vitro usually results from the use of peptide at a high concentration,
generating a high level of specific peptide/MHC complexes on antigen
presenting cells (APCs), which will effectively activate these CTL
(Alexander-Miller et al., Proc. Natl. Acad. Sci. USA 93: 4102-7 (1996)).

[0016] Although advances have been made in the development of
molecular-targeting drugs for cancer therapy, the ranges of tumor types
that respond as well as the effectiveness of the treatments are still
very limited. Hence, it is urgent to develop new anti-cancer agents that
target molecules highly specific to malignant cells and are likely to
cause minimal or no adverse reactions. To achieve the goal molecules
whose physiological mechanisms are well defined need to be identified. A
powerful strategy toward these ends would combine screening of
up-regulated genes in cancer cells on the basis of genetic information
obtained on cDNA microarrays with high-throughput screening of their
effect on cell growth, by inducing loss-of-function phenotypes with RNAi
systems (Kikuchi, T. et al., Oncogene 22: 2192-2205 (2003)).

SUMMARY OF THE INVENTION

[0017] The present invention features a method of diagnosing or
determining a predisposition to non-small cell lung cancer (NSCLC) in a
subject by determining an expression level of a non-small cell lung
cancer-associated gene that is selected from the group of KIF11, GHSR1b,
NTSR1, and FOXM1 in a patient derived biological sample. An increase of
the expression level of any of the genes compared to a normal control
level of the genes indicates that the subject suffers from or is at risk
of developing NSCLC.

[0018] The invention also provides methods of providing a prognosis of a
patient diagnosed with NSCLC. In particular, the methods involve
detecting expression of KOC1, KIF11, or KOC1 in combination with
expression of KIF11.

[0019] A "normal control level" indicates an expression level of any of
the genes detected in a normal, healthy individual or in a population of
individuals known not to be suffering from NSCLC. A control level is a
single expression pattern derived from a single reference population or
from a plurality of expression patterns. In contrast to a "normal control
level", the "control level" is an expression level of the gene detected
in an individual or a population of individuals whose background of the
disease state is known (i.e., cancerous or non-cancerous). Thus, the
control level may be determined base on the expression level of the gene
in a normal, healthy individual, in a population of individuals known not
to be suffering from NSCLC, a patient of NSCLC or a population of the
patients. The control level corresponding to the expression level of the
gene in a patient of non-small cell lung cancer or a population of the
patients is referred to as "NSCLC control level". Furthermore, the
control level can be a database of expression patterns from previously
tested cells.

[0020] An increase in the expression level of any one of the genes of
KIF11, GHSR1b, NTSR1, and FOXM1 detected in a test biological sample
compared to a normal control level indicates that the subject (from which
the sample was obtained) suffers from NSCLC. Alternatively, the
expression level of any one or all of the genes in a biological sample
may be compared to an NSCLC control level of the same gene(s).

[0021] Gene expression is increased or decreased 10%, 25%, 50% or more
compared to the control level. Alternatively, gene expression is
increased or decreased 1, 2, 5 or more fold compared to the control
level. Expression is determined by detecting hybridization, e.g., on a
chip or an array, of an NSCLC gene probe to a gene transcript of a
patient-derived biological sample. The patient-derived biological sample
may be any sample derived from a subject, e.g., a patient known to or
suspected of having NSCLC. For example, the biological sample may be
tissue containing sputum, blood, serum, plasma or lung cell.

[0022] The invention also provides a non-small cell lung cancer reference
expression profile comprising a pattern of gene expression levels of two
or more genes selected from the group of KIF11, GHSR1b, NTSR1, and FOXM1.

[0023] The invention also provides a kit comprising two or more detection
reagents which detects the expression of one or more of genes selected
from the group of KIF11, GHSR1b, NTSR1, and FOXM1 (e.g., via detecting
mRNA and polypeptide). Also provided is an array of polynucleotides that
binds to one or more of the genes selected from the group of KIF11,
GHSR1b, NTSR1, and FOXM1. The kits of the invention may also comprise
reagents used to detect the expression of KIF11 and KOC1 to be used for
the prognosis of NSCLC. The invention also provides kits for the
detection of compounds that regulate RNA transporting activity. The kits
may comprise a cell expressing a KIF11 polypeptide, or functional
equivalent, a KOC1 polypeptide, or functional equivalent, and RNA to be
transported, and DCTN1. The kits of the invention may also be used to
screen for compounds for treating or preventing NSCLC. The kits may
comprise a KOC1 polypeptide, or functional equivalent, and an RNA that is
bound by the KOC1 polypeptide or functional equivalent.

[0024] The invention further provides methods of identifying compounds
that inhibit the expression level of an NSCLC-associated gene (KIF11,
GHSR1b, NTSR1 or FOXM1) by contacting a test cell expressing an
NSCLC-associated gene with a test compound and determining the expression
level of the NSCLC-associated gene. The test cell may be an NSCLC cell. A
decrease of the expression level compared to a normal control level of
the gene indicates that the test compound is an inhibitor of the
expression or function of the NSCLC-associated gene. Therefore, if a
compound suppresses the expression level of KIF11, GHSR1b, NTSR1 or FOXM1
compared to a control level, the compound is expected to reduce a symptom
of NSCLC.

[0025] Alternatively, the present invention provides a method of screening
for a compound for treating or preventing NSCLC. The method includes
contacting a polypeptide selected from the group of KIF11, GHSR1b, NTSR1,
and FOXM1 with a test compound, and selecting the test compound that
binds to or suppresses the biological activity of the polypeptide. The
invention further provides a method of screening for a compound for
treating or preventing NSCLC, which includes the steps of contacting a
test compound with a cell that expresses KIF11, GHSR1b, NTSR1 or FOXM1
protein or introduced with a vector comprising the transcriptional
regulatory region of KIF11, GHSR1b, NTSR1 or FOXM1 gene upstream of a
reporter gene, and then selecting the test compound that reduces the
expression level of the KIF11, GHSR1b, NTSR1 or FOXM1 protein or protein
encoded by the reporter gene. According to these screening methods, the
test compound that suppresses the biological activity or the expression
level compared to a control level is expected to reduce a symptom of
NSCLC. Furthermore, the present invention provides a method of screening
for a compound for treating or preventing NSCLC wherein the binding
between KIF11 and KOC1, or GHSR1b or NTSR1 and NMU is detected. Compounds
that inhibit the binding between KIF11 and KOC1, or GHSR1b or NTSR1 and
NMU are expected to reduce a symptom of NSCLC.

[0026] We detected a novel intra-cellular and inter-cellular
RNA-transporting system in lung carcinomas, involving transactivation of
KOC1 and KIF11. A complex of these two molecules in lung tumors was able
to bind mRNAs encoding proteins known to function in intercellular
adhesion, cancer-cell progression, and oncogenesis, and transport them to
neighboring cells through ultrafine intercellular structures. In
particular, evidence provided here shows that KOC1 binds to KIF11 at the
RRM domain in the N-terminal region of KOC1. In addition, evidence
provided here shows inhibition of their binding by dominant-negative KOC1
mutants effectively suppressed growth of NSCLC cells in vitro. For
example, KOC1 fragments (or nucleic acids encoding them) comprising the
RRM domains of KOC1 can be used as dominant negative fragments to
suppress cell proliferation and thus treat cancer. Alternatively, the
KOC1 fragment may comprise the ribonucleoprotein K-homologous (KH)
domain.

[0027] The invention also provides methods of identifying polypeptides and
other compounds that modulate RNA transport activity. For example, a
polypeptide can be tested for RNA transporting activity by contacting the
polypeptide with a KIF11 polypeptide or a functional equivalent thereof
with an RNA that can be transported by KIF11 under conditions suitable
for transportation of RNA. Alternatively, agents that modulate RNA
transporting activity can be tested by contacting a test agent with a
KIF11 polypeptide or a functional equivalent thereof with an RNA that can
be transported by KIF11 under conditions suitable for transportation of
RNA. Test agents useful for treating NSCLC by testing the agents for the
ability to inhibit binding between a KOC1 polypeptide, or a functional
equivalent, and an RNA that is bound by KOC1 or the complex of KOC1 and
KIF11.

[0028] Immunohistochemical analysis of lung-cancer tissue microarrays
demonstrated that transactivation of KOC1 and KIF11 was significantly
associated with poor prognosis of lung-cancer patients.

[0029] Methods for treating or preventing NSCLC and compositions to be
used for such methods are also provided. Therapeutic methods include a
method of treating or preventing NSCLC in a subject by administering to
the subject a composition of an antisense, short interfering RNA (siRNA)
or a ribozyme that reduce the expression of KIF11, GHSR1b, NTSR1 or FOXM1
gene, or a composition comprising an antibody or fragment thereof that
binds and suppresses the function of a polypeptide encoded by the gene.
The compositions of the invention may also comprise a dominant negative
KOC1 mutant (or nucleic acids encoding it) comprising a KOC1 fragment
that contains one or more RRM domains and/or KH domains of KOC1.

[0030] The invention also includes vaccines and vaccination methods. For
example, a method of treating or preventing NSCLC in a subject is carried
out by administering to the subject a vaccine containing a polypeptide
encoded by KIF11, GHSR1b, NTSR1 or FOXM1 gene, or an immunologically
active fragment of the polypeptide. An immunologically active fragment is
a polypeptide that is shorter in length than the full-length
naturally-occurring protein and which induces an immune response upon
introduction into the body. For example, an immunologically active
fragment includes a polypeptide of at least 8 residues in length that
stimulates an immune cell such as a T cell or a B cell in vivo. Immune
cell stimulation can be measured by detecting cell proliferation,
elaboration of cytokines (e.g., IL-2) or production of antibody.

[0031] Other therapeutic methods include those wherein a compound selected
by the screening method of the present invention is administered.

[0032] Also included in the invention are double-stranded molecules that
comprise a sense strand and an antisense strand. The sense strand
comprises a ribonucleotide sequence corresponding to a target sequence
comprised within the mRNA of a KIF11, GHSR1b, NTSR1 or FOXM1 gene, and
the antisense strand is a complementary sequence to the sense strand.
Such double-stranded molecules of the present invention can be used as
siRNAs against KIF11, GHSR1b, NTSR1 or FOXM1 gene. Furthermore, the
present invention relates to vectors encoding the double-stranded
molecules of the present invention.

[0033] The present application also provides a composition for treating
and/or preventing NSCLC using any of the antisense polynucleotides or
siRNAs against KIF11, GHSR1b, NTSR1 or FOXM1 gene, or an antibody that
binds to a polypeptide encoded by KIF11, GHSR1b, NTSR1 or FOXM1 gene.
Other compositions include those that contain a compound selected by the
screening method of the present invention as an active ingredient.

[0034] It is to be understood that both the foregoing summary of the
invention and the following detailed description are of a preferred
embodiment, and not restrictive of the invention or other alternate
embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1 shows photographs confirming the relationship between KOC1
and KIF11. [0036] (a) depicts the result of co-immunoprecipitation of
KOC1 and KIF11 confirming the interaction between KOC1 and KIF11. A549
cells were transiently co-transfected with Flag-tagged KIF11 and
myc-tagged KOC1, immunoprecipitated with anti-Flag M2 agarose, and
subsequently immunoblotted with anti-myc antibody. In contrast, using the
same combination of vectors and cells, the cells were immunoprecipitated
with anti-myc agarose and immunoblotted with anti-Flag M2 antibody. A
band corresponding to the immunoblotted protein was found only when both
constructs were co-transfected.

[0037] (b) depicts the result of immunocytochemical staining showing the
co-localization of KOC1 and KIF11. COS-7 cells were transiently
transfected with FLAG-tagged KIF11 and myc-tagged KOC1, and their
co-localization was detected mainly in the cytoplasm using FITC-labeled
anti-FLAG antibody and rhomamine-labeled anti-myc antibody.

[0038] (c) depicts the result of reciprocal co-immunoprecipitation of
endogenous KOC1 and KIF11 from extracts of lung-cancer cell lines A549
and LC319. (upper panel) Western-blot analysis of both cell extracts
immunoprecipitated with anti-KOC1 antibodies, with KIF11 protein detected
in the immunoprecipitate. (lower panel) Western-blot of extracts
immunoprecipitated with anti-KIF11 antibodies, with KOC1 protein detected
in the immunoprecipitate.

[0040] (a) depicts the result of QRT-PCR examining expression of KOC1 and
KIF11 in clinical samples of NSCLC and corresponding normal lung tissues.
Y-axis indicates the relative expression rate of the two genes (KOC1 or
KIF11/ACTS).

[0041] (b) depicts the result of QRT-PCR examining expression of KOC1 and
KIF11 among 20 lung-cancer cell lines.

[0042] (c) depicts the result of Northern-blot analysis detecting
expression of KOC1 and KIF11 in normal human tissues.

[0044] (a) shows schematic drawing of five KOC1 deletion mutants lacking
either or both of the terminal regions, with N- and C-terminals tagged
with FLAG and HA respectively. KH, ribonucleoprotein K-homologous domain.

[0045] (b) depicts the result of immunoprecipitation experiments for
identification of the region of KOC1 that binds to KIF11. The KOC1DEL4
and KOC1DEL5 constructs, which lacked two RNA-recognition motifs, (RRM)
did not retain any appreciable ability to interact with endogenous KIF11.

[0047] (a) depicts the result of Western blotting with immunoprecipitated
KOC1 deletion mutants and DIG-labeled RAB35 full length mRNA for
identification of the mRNA-binding region in KOC1.

[0048] (b) depicts the result of Northwestern with immunoprecipitated KOC1
deletion mutants and DIG-labeled RAB35 full length mRNA for
identification of the mRNA-binding region in KOC1. The KOC1DEL3 and
KOC1DEL5, did not bind to any of these mRNAs, and the KOC1DEL4, which is
a construct with the four KH domains only, showed similar binding
affinities for mRNAs to the KOC1DEL2, a construct without C-terminal two
KH domains.

[0052] (b) are photographs showing transport of KOC1-RAB35 mRNA RNP
complex from one COS-7 cell that contains a high level of KOC1-RNP
complex (cell A) to another cell with a lower level of the complex
(stained simply with CellTracker; cell B). Small particles of KOC1-RAB35
mRNA complex as well as KOC1 particles were transferred from cell A to
cell B through ultrafine intercellular structures (arrows).

[0060] (e) are photographs showing that no significant difference in the
protein level of RAB35-EGFP fused-protein was found between COS-7 cells
that were co-transfected with RAB35-EGFP and HA-tagged-KOC1 vectors, and
those with RAB35-EGFP and mock plasmid vectors. This indicates that KOC1
is not likely to interfere with translation of RAB35-EGFP mRNA.

[0061] FIG. 8 shows the effect of KIF11 siRNAs on cells.

[0062] (a) depicts the inhibition on the growth of NSCLC cells by siRNAs
against KIF11. The expression of KIF11 in response to specific siRNAs
(si-KIF#1, #2, and #3) or control siRNAs (EGFP, LUC, SC) in A549 cells,
was analyzed by semiquantitative RT-PCR.

[0076] (b) depicts the results of Kaplan-Meier analysis of tumor-specific
survival times according to KOC1 expression (left panel) and KIF11
expression (right panel) on tissue microarrays.

[0077] FIG. 12 is schematic model for the mechanism of intracellular and
cell-to-cell mRNA transport by KOC1-KIF11-DCTN1 complexes on
microtubules. The KOC1 ribonucleoprotein complex, including KIF11
motor-protein and DCTN1, transports KOC1-associated mRNAs through the
structure of microtubules within or between mammalian somatic cells. This
model implies that proliferating cancer-cells may communicate actively by
engaging this molecular complex in a system that transports mRNAs
critical for cancer growth or progression from one cell to another FIG. 8
shows the relationship between NMU and GHSR1b/NTSR1.

[0078] FIG. 13 (a) shows the result of semiquantitative RT-PCR analysis
depicting the expression of NMU, candidate receptors, and their known
ligands detected in NSCLC cell lines.

[0079] (b) shows GHSR1b expression in normal human tissues.

[0080] (c) depicts the result of immunocytochemical staining using
FITC-labeled anti-FLAG antibody showing the co-localization of NMU and
GHSR1b/NTSR1 on the cell surface of COS-7 cells that were transiently
transfected with FLAG-tagged GHSR1b or NTSR1.

[0081] (d) depicts the interaction of NMU with GHSR1b/NTSR1. COS-7 cells
were transiently transfected with the same vectors, and binding of
rhodamine-labeled NMU-25 to the cell surface was detected by flow
cytometry. As negative controls for these assays, three ligand/cell
combinations were prepared: 1) non-transfected COS-7 cells; 2)
NMU-25-rhodamine vs non-transfected COS-7 cells; and 3) COS-7 cells
transfected only with GHSR1b or NTSR1.

[0082] (e) depicts the results of receptor-ligand binding assay using the
LC319 and PC-14 cells treated with NMU-25.

[0083] (f) depicts cAMP production of NMU-treated NSCLC cells.

[0084] FIG. 14 shows the effect of siRNAs on cells.

[0085] (a) depicts the inhibition on the growth of NSCLC cells by siRNAs
against GHSR1b and NTSR1. Expression of GHSR or NTSR1 in response to
specific siRNAs (si-GHSR or si-NTSR1) or control siRNAs (EGFP, LUC, SCR)
in A549 and LC319 cells were analyzed by semiquantitative RT-PCR.

[0086] (b) depicts the result of triplicate MTT assays evaluating
viability of A549 or LC319 cells in response to si-GHSR, NTSR1, EGFP,
LUC, or SCR.

[0090] (c) depicts the result of semiquantitative RT-PCR using mRNAs from
LC319 cells incubated with NMU-25 or BSA (control) (100 μM) detecting
induction of FOXM1 as the candidate downstream target gene of NMU.

[0094] FIG. 17 is a schematic model for promotion of cancer cell growth
and invasion through the NMU-receptor interaction in the autocrine
NMU-GHSR1b oncogenic signaling pathway. Binding of NMU to GHSR1b and/or
NTSR1 leads to the activation of adenylate cyclase, accumulation of
intracellular cAMP and following activation of cAMP-dependent protein
kinase (PKA). The release of catalytic subunits of PKA (C) from the
regulatory subunits (R) is resulting in the activation of downstream
FOXM1 gene and/or related target genes.

[0096] To investigate the mechanisms of lung carcinogenesis and identify
genes that might be useful as diagnostic markers or targets for
development of new molecular therapies, genes specifically up-regulated
in non-small cell lung cancers (NSCLC) were searched by means of cDNA
microarray. Through the analysis, a couple of candidate therapeutic
target genes were identified. Two genes, KH domain containing protein
over-expressed in cancer (KOC1) and neuromedin U (NMU) were abundantly
expressed in clinical NSCLC samples as well as NSCLC cell lines examined.
However, their expression was hardly detectable in corresponding
non-cancerous lung tissue. The growth of NSCLC cells that over-expressed
endogenous NMU was significantly inhibited by anti-NMU antibody.
Furthermore, the treatment of NSCLC cells with siRNA against KOC1 and/or
NMU suppressed the expression of the gene and resulted in growth
inhibition of the NSCLC cells. Furthermore, KOC1 was identified to bind
to kinesin family member 11 (KIF11) of the cancer cells, whereas NMU
bound to the neuropeptide G protein-coupled receptors (GPCRs), growth
hormone secretagogue receptor 1b (GHSR1b) and neurotensin receptor 1
(NTSR1). NMU ligand-receptor system was identified to activate Homo
sapiens forkhead box M1 (FOXM1). Interestingly, GHSR1b, NTSR1, FOXM1, and
KIF11 were all specifically over-expressed in NSCLC cells.

[0097] RNA binding protein KOC1 and microtubles motor protein KIF11 is
required for the localization of some kinds of mRNA needed in
embryogenesis and carcinogenesis (FIG. 12). As previously reported by the
present inventors, treatment of NSCLC cells with specific siRNA to reduce
expression of KOC1 resulted in growth suppression. In this study, KIF11
was demonstrated to associate with KOC1 in NSCLC cells and to be the
target for the growth-promoting effect of KOC1 in lung tumors. The
present inventors revealed that KOC1 not only co-localized with KIF11 in
human normal tissues, NSCLCs, and cell lines, but also directly
interacted with KIF11 in NSCLC cells in vitro, and that the treatment of
NSCLC cells with siRNAs for KIF11 reduced its expression and led to
growth suppression. The results show that KOC1-KIF11 signaling affects
growth of NSCLC cells. As shown below, dominant negative fragments of
KOC1 (e.g., those containing the RRM domains) can be used to inhibit
proliferation of cancer cells. By expression analysis, increased
expression of KOC1 and KIF11 was detected in the majority of NSCLC
samples, but not in normal lung tissues. Since most of the clinical NSCLC
samples used for the present analysis were at an early and operable
stage, KOC1 and KIF11 can be conveniently used as a biomarker for
diagnosing early-stage lung cancer, in combination with fiberscopic
transbronchial biopsy (TBB) or sputum cytology.

[0098] Therefore, KOC1 and KIF11 are essential for an oncogenic pathway in
NSCLCs. The data reported here provide evidence for designing new
anti-cancer drugs, specific for lung cancer, which target the KOC1-KIF11
pathway. They also show that siRNAs can be used to treat
chemotherapy-resistant, advanced lung cancers.

[0099] A significant increase in the sub-G1 fraction of NSCLC cells
transfected with siRNA-NMU suggested that blocking the autocrine
NMU-signaling pathway could induce apoptosis. The present inventors also
found other evidence supporting the significance of this pathway in
carcinogenesis; e.g., addition of NMU into the medium promoted the growth
of COS-7 cells in a dose-dependent manner, and addition of anti-NMU
antibody into the culture medium inhibited this NMU-enhanced cell growth,
possibly by neutralizing NMU activity. Moreover, the growth of NSCLC
cells that endogenously over-expressed NMU was significantly inhibited by
anti-NMU antibody. The expression of NMU also resulted in significant
promotion of COS-7 cell invasion in in vitro assays. These results show
that NMU is an important growth factor for NSCLC and is associated with
cancer cell invasion, functioning in an autocrine manner, and that
screening molecules targeting the NMU-receptor growth-promoting pathway
is a useful therapeutic approach for treating NSCLCs. By
immunohistochemical analysis, increased expression of NMU protein was
detected in the majority of NSCLC (SCC, ADC, LCC, and BAC) and SCLC
samples, but not in normal lung tissues. Since NMU is a secreted protein
and most of the clinical NSCLC samples used for the present analysis were
at an early and operable stage, NMU can be conveniently used as a
biomarker for diagnosis of early-stage lung cancer, in combination with
fiberscopic transbronchial biopsy (TBB), sputum cytology, or blood tests.

[0100] Two receptors, NMU1R (FM3/GPR66) and NMU2R (FM4) are known to
interact with NMU. The results presented here, however, indicated that
these two known receptors were not the targets for the autocrine
NMU-signaling pathway in NSCLCs; instead, GHSR1b and NTSR1 proved to be
the targets for the growth-promoting effect of NMU in lung tumors. The
present inventors revealed that NMU-25 bound to these receptors on the
cell surface, and that treatment of NSCLC cells with siRNAs for GHSR1b or
NTSR1 reduced expression of the receptors and led to apoptosis. The
results show that NMU affects growth of NSCLC cells by acting through
GHSR1b and/or NTSR1 (FIG. 14). GHSR is a known receptor of Ghrelin
(GHRL), a recently identified 28-amino-acid peptide capable of
stimulating release of pituitary growth hormone and appetite in humans
(Lambert, P. D. et al., Proc. Natl. Acad. Sci. 98: 4652-4657 (2001);
Petersenn, S. et al., Endocrinology 142: 2649-2659 (2001); Kim K. et al.,
Clin. Endocrinol. 54: 705-860 (2001); Kojima, M. et al., Nature 402:
656-660 (1999)). Of the two transcripts known to be receptors for GHRL,
GHSR1a and GHSR1b, over-expression of only GHSR1b was detected in NSCLC
tissues and cell lines. Since GHRL was not expressed in the NSCLCs
examined, GHSR1b was suspected to have a growth-promoting function in
lung tumors through binding to NMU, but not to GHRL.

[0101] NTSR1 is one of three receptors of neurotensin (NTS), a brain and
gastrointestinal peptide that fulfills many central and peripheral
functions (Heasley, L. E. Oncogene 20: 1563-1569 (2001)). NTS modulates
transmission of dopamine and secretion of pituitary hormones, and exerts
hypothermic and analgesic effects in the brain while it functions as a
peripheral hormone in the digestive tract and cardiovascular system.
Others have reported that NTS is produced and secreted in several human
cancers, including small-cell lung cancers (SCLC) (Heasley, L. E.
Oncogene 20: 1563-1569 (2001)). The expression of NTS was detected in
four of the 15 NSCLC cell lines that were examined in the present
invention (FIG. 13a), but the expression pattern of NTS was not
necessarily concordant with that of NMU or NTSR1. Therefore NTS may,
along with NMU, contribute to the growth of NSCLC through NTSR1 or other
receptor(s) in a small subset of NSCLCs. In the present experiments the
majority of the cancer cell lines and clinical NSCLCs that expressed NMU
also expressed GHSR1b and/or NTSR1, indicating that these ligand-receptor
interactions were involved in a pathway that is central to the
growth-promoting activity of NMU in NSCLCs.

[0102] NMU signaling pathway affects the growth promotion of lung-cancer
cells by transactivating a set of downstream genes including FOXM1. FOXM1
was known to be over-expressed in several types of human cancers (Teh, M.
T. et al., Cancer Res. 62, 4773-4780; van den Boom, J. et al., (2003).
Am. J. Pathol. 163, 1033-1043; Kalinichenko, V. V. et al., (2004). Genes.
Dev. 18, 830-850). The "forkhead" gene family, originally identified in
Drosophila, comprises transcription factors with a conserved 100-amino
acid DNA-binding motif, and has been shown to play important roles in
regulating the expression of genes involved in cell growth,
proliferation, differentiation, longevity, and transformation.
Cotransfection assays in the human hepatoma HepG2 cell line demonstrated
that FOXM1 protein stimulated expression of both the cyclin B1 (CCNB1)
and cyclin D1 (CCND1) (Wang, X. et al., (2002). Proc. Nat. Acad. Sci. 99,
16881-16886.), suggesting that these cyclin genes are direct FOXM1
transcription targets and that FOXM1 controls the transcription network
of genes that are essential for cell division and exit from mitosis. It
should be noted that we observed activation of CCNB1 in the majority of a
series of NSCLC and its good concordance of the expression to FOXM1 (data
not shown). The promotion of cell growth in NSCLC cells by NMU might
reflect transactivation of FOXM1, which would affect the function of
those molecular pathways in consequence. Therefore, NMU, two newly
revealed receptors for this molecule, GHSR1b and NTSR1, and their
downstream gene FOXM1 are involved in an autocrine growth-promoting
pathway in NSCLCs. The data reported here provide the basis for designing
new anti-cancer drugs, specific for lung cancer, that target the
NMU-GHSR1b/NTSR1-FOXM1 pathway. They also show that siRNAs that interfere
with this pathway can be used to treat chemotherapy-resistant, advanced
lung cancers.

[0103] These data show that KOC1-KIF11 signaling pathway is frequently
up-regulated in lung carcinogenesis, and that NMU an important autocrine
growth factor for NSCLC, acting through GHSR1b and NTSR1 receptor
molecules. Thus, selective suppression of components of these complexes
can suppress the development and/or progression of lung carcinogenesis
and targeting these pathways are conveniently used in therapeutic and
diagnostic strategies for the treatment of lung-cancer patients.

Diagnosing Non-Small Cell Lung Cancer (NSCLC)

[0104] By measuring the expression level of KIF11, GHSR1b, NTSR1 or FOXM1
gene in a biological derived from a subject, the occurrence of NSCLC or a
predisposition to develop NSCLC in the subject can be determined. The
invention involves determining (e.g., measuring) the expression level of
at least one, and up to all of KIF11, GHSR1b, NTSR1, and FOXM1 gene in
the biological sample.

[0105] According to the present invention, a gene transcript of
NSCLC-associated gene, KIF11, GHSR1b, NTSR1 or FOXM1, is detected for
determining the expression level of the gene. The expression level of a
gene can be detected by detecting the expression products of the gene,
including both transcriptional and translational products, such as mRNA
and proteins. Based on the sequence information provided by the
GenBank® database entries for the known sequences, KIF11
(NM--004523), GHSR1b (NM--004122), NTSR1 (NM--002531), and
FOXM1 (No. NM--202003) genes can be detected and measured using
techniques well known to one of ordinary skill in the art. The nucleotide
sequences of the KIF11, GHSR1b, NTSR1, and FOXM1 genes are described as
SEQ ID NOs: 1, 3, 5, and 106, respectively, and the amino acid sequences
of the proteins encoded by the genes are described as SEQ ID NOs: 2, 4,
6, and 107.

[0106] For example, sequences within the sequence database entries
corresponding to KIF11, GHSR1b, NTSR1 or FOXM1 gene can be used to
construct probes for detecting their mRNAs by, e.g., Northern blot
hybridization analysis. The hybridization of the probe to a gene
transcript in a subject biological sample can be also carried out on a
DNA array. The use of an array is preferred for detecting the expression
level of a plurality of the NSC genes (KIF11, GHSR1b, NTSR1, and FOXM1).
As another example, the sequences can be used to construct primers for
specifically amplifying KIF11, GHSR1b, NTSR1 or FOXM1 gene in, e.g.,
amplification-based detection methods such as reverse-transcription based
polymerase chain reaction (RT-PCR). Furthermore, the expression level of
KIF11, GHSR1b, NTSR1 or FOXM1 gene can be analyzed based on the quantity
of the expressed proteins encoded by the gene. A method for determining
the quantity of the expressed protein includes immunoassay methods.
Alternatively, the expression level of KIF11, GHSR1b, NTSR1 or FOXM1 gene
can also be determined based on the biological activity of the expressed
protein encoded by the gene. For example, a protein encoded by KIF11 gene
is known to bind to KOC1, and thus the expression level of the gene can
be detected by measuring the binding ability to KOC1 due to the expressed
protein. Furthermore, KIF11 protein is known to have a cell proliferating
activity. Therefore, the expression level of KIF11 gene can be determined
using such cell proliferating activity as an index. On the other hand
GHSR1b and NTSR1 proteins are known to bind to NMU, and also have a cell
proliferating activity. Thus, similarly to KIF11, the expression levels
of GHSR1b and NTSR1 genes can be detected by measuring their binding
ability to NMU or cell proliferating activity due to the expressed
protein.

[0107] Any biological materials may be used as the biological sample for
determining the expression level so long as any of the KIF11, GHSR1b,
NTSR1, and FOXM1 genes can be detected in the sample and includes test
cell populations (i.e., subject derived tissue sample). Preferably, the
biological sample comprises a lung cell (a cell obtained from the lung).
Gene expression may also be measured in blood, serum or other bodily
fluids such as sputum. Furthermore, the test sample may be cells purified
from a tissue.

[0108] The subject diagnosed for NSCLC according to the method is
preferably a mammal and includes human, non-human primate, mouse, rat,
dog, cat, horse and cow.

[0109] The expression level of one or more of KIF11, GHSR1b, NTSR1 or
FOXM1 gene in the biological sample is compared to the expression
level(s) of the same genes in a reference sample. The reference sample
includes one or more cells with known parameters, i.e., cancerous or
non-cancerous. The reference sample should be derived from a tissue type
similar to that of the test sample. Alternatively, the control expression
level may be determined based on a database of molecular information
derived from cells for which the assayed parameter or condition is known.

[0110] Whether or not a pattern of the gene expression levels in a
biological sample indicates the presence of NSCLC depends upon the
composition of the reference cell population. For example, when the
reference cell population is composed of non-cancerous cells, a similar
gene expression level in the test biological sample to that of the
reference indicates that the test biological sample is non-cancerous. On
the other hand, when the reference cell population is composed of
cancerous cells, a similar gene expression profile in the biological
sample to that of the reference indicates that the test biological sample
includes cancerous cells.

[0111] The test biological sample may be compared to multiple reference
samples. Each of the multiple reference samples may differ in the known
parameter. Thus, a test sample may be compared to a reference sample
known to contain, e.g., NSCLC cells, and at the same time to a second
reference sample known to contain, e.g., non-NSCLC cells (normal cells).

[0112] According to the invention, the expression of one or more of the
NSCLC-associated genes, KIF11, GHSR1b, NTSR1, and FOXM1, is determined in
the biological sample and compared to the normal control level of the
same gene. The phrase "normal control level" refers to an expression
profile of KIF11, GHSR1b, NTSR1 or FOXM1 gene typically found in a
biological sample derived from a population not suffering from NSCLC. The
expression level of KIF11, GHSR1b, NTSR1 or FOXM1 gene in the biological
samples from a control and test subjects may be determined at the same
time or the normal control level may be determined by a statistical
method based on the results obtained by analyzing the expression level of
the gene in samples previously collected from a control group. An
increase of the expression level of KIF11, GHSR1b, NTSR1 or FOXM1 gene in
the biological sample derived from a patient derived tissue sample
indicates that the subject is suffering from or is at risk of developing
NSCLC.

[0113] An expression level of KIF11, GHSR1b, NTSR1 or FOXM1 gene in a test
biological sample can be considered altered when the expression level
differs from that of the reference by more than 1.0, 1.5, 2.0, 5.0, 10.0
or more fold. Alternatively, an expression level of KIF11, GHSR1b, NTSR1
or FOXM1 gene in a test biological sample can be considered altered, when
the expression level is increased or decreased to that of the reference
at least 50%, 60%, 80%, 90% or more.

[0114] The difference in gene expression between the test sample and a
reference sample may be normalized to a control, e.g., housekeeping gene.
For example, a control polynucleotide includes those whose expression
levels are known not to differ between the cancerous and non-cancerous
cells. The expression levels of the control polynucleotide in the test
and reference samples can be used to normalize the expression levels
detected for KIF11, GHSR1b, NTSR1 or FOXM1 gene. The control genes to be
used in the present invention include β-actin, glyceraldehyde
3-phosphate dehydrogenase and ribosomal protein P1.

[0115] The differentially expressed KIF11, GHSR1b, NTSR1 or FOXM1 gene
identified herein also allow for monitoring the course of treatment of
NSCLC. In this method, a test biological sample is provided from a
subject undergoing treatment for NSCLC. If desired, multiple test
biological samples are obtained from the subject at various time points
before, during or after the treatment. The expression of one or more of
KIF11, GHSR1b, NTSR1 or FOXM1 gene in the sample is then determined and
compared to a reference sample with a known state of NSCLC that has not
been exposed to the treatment.

[0116] If the reference sample contains no NSCLC cells, a similarity in
the expression level of KIF11, GHSR1b, NTSR1 or FOXM1 gene in the test
biological sample and the reference sample indicates the efficaciousness
of the treatment. However, a difference in the expression level of KIF11,
GHSR1b, NTSR1 or FOXM1 gene in the test and the reference samples
indicates a less favorable clinical outcome or prognosis. In particular,
increased expression of KOC1, KIF11, or KOC1 in combination with
increased expression of KIF11 is significantly associated with poor
prognosis.

[0117] The term "efficacious" refers that the treatment leads to a
reduction in the expression of a pathologically up-regulated gene
(including the present indicator genes, KIF11, GHSR1b, NTSR1, and FOXM1),
or a decrease in size, prevalence or metastatic potential of NSCLC in a
subject. When a treatment is applied prophylactically, "efficacious"
means that the treatment retards or prevents occurrence of NSCLC or
alleviates a clinical symptom of NSCLC. The assessment of NSCLC can be
made using standard clinical protocols. Furthermore, the efficaciousness
of a treatment is determined in association with any known method for
diagnosing or treating NSCLC. For example, NSCLC is diagnosed
histopathologically or by identifying symptomatic anomalies such as
chronic cough, hoarseness, coughing up blood, weight loss, loss of
appetite, shortness of breath, wheezing, repeated bouts of bronchitis or
pneumonia and chest pain.

[0118] Moreover, the present method for diagnosing NSCLC may also be
applied for assessing the prognosis of a patient with the cancer by
comparing the expression level of KIF11, KOC1, GHSR1b, NTSR1, FOXM1 gene,
or a combination thereof (e.g., KOC1 and KIF11) in the patient-derived
biological sample. Alternatively, the expression level of the gene(s) in
the biological sample may be measured over a spectrum of disease stages
to assess the prognosis of the patient.

[0119] An increase in the expression level of KIF11, KOC1, GHSR1b, NTSR1
or FOXM1 gene compared to a normal control level indicates less favorable
prognosis. A similarity in the expression level of KIF11, KOC1, GHSR1b,
NTSR1 or FOXM1 gene compared to a normal control level indicates a more
favorable prognosis of the patient. Preferably, the prognosis of a
subject can be assessed by comparing the expression profile of KIF11,
KOC1, GHSR1b, NTSR1 or FOXM1 gene. In some embodiments, expression levels
of KIF11 and KOC1 are determined.

Expression Profile

[0120] The invention also provides an NSCLC reference expression profile
comprising a pattern of gene expression levels of two or more of KIF11,
KOC1, GHSR1b, NTSR1 and FOXM1 genes. The expression profile serves as a
control for the diagnosis of NSCLC or predisposition for developing the
disease, monitoring the course of treatment and assessing prognosis of a
subject with the disease.

Kits of the Invention

[0121] The invention also provides a kit comprising two or more detection
reagents, e.g., a nucleic acid that specifically binds to or identifies
one or more of KIF11, KOC1, GHSR1b, NTSR1 and FOXM1 genes. Such nucleic
acids specifically binding to or identifying one or more of KIF11, KOC1,
GHSR1b, NTSR1 and FOXM1 genes are exemplified by oligonucleotide
sequences that are complementary to a portion of KIF11, KOC1, GHSR1b,
NTSR1 or FOXM1 polynucleotides or antibodies which bind to polypeptides
encoded by the KIF11, KOC1, GHSR1b, NTSR1 or FOXM1 gene. The reagents are
packaged together in the form of a kit. The reagents, such as a nucleic
acid or antibody (either bound to a solid matrix or packaged separately
with reagents for binding them to the matrix), a control reagent
(positive and/or negative) and/or a means of detection of the nucleic
acid or antibody are preferably packaged in separate containers.
Instructions (e.g., written, tape, VCR, CD-ROM, etc.) for carrying out
the assay may be included in the kit. The assay format of the kit may be
Northern hybridization or sandwich ELISA known in the art.

[0122] For example, a detection reagent is immobilized on a solid matrix
such as a porous strip to form at least one detection site. The
measurement or detection region of the porous strip may include a
plurality of detection sites, each detection site containing a detection
reagent. A test strip may also contain sites for negative and/or positive
controls. Alternatively, control site(s) is located on a separate strip
from the test strip. Optionally, the different detection sites may
contain different amounts of immobilized reagents, i.e., a higher amount
in the first detection site and lesser amounts in subsequent sites. Upon
the addition of a test biological sample, the number of sites displaying
a detectable signal provides a quantitative indication of the amount of
KIF11, GHSR1b, NTSR1 or FOXM1 gene, or polypeptides encoded by the gene
present in the sample. The detection sites may be configured in any
suitably detectable shape and are typically in the shape of a bar or dot
spanning the width of a teststrip.

[0123] Alternatively, the kit contains a nucleic acid substrate array
comprising two or more of the KIF11, GHSR1b, NTSR1, and FOXM1 gene
sequences. The expression of 2 or 3 of the genes represented by KIF11,
GHSR1b, NTSR1, and FOXM1 genes are identified by virtue of the level of
binding to an array test strip or chip. The substrate array can be on,
e.g., a solid substrate, e.g., a "chip" as described in U.S. Pat. No.
5,744,305.

[0124] In some embodiments, the kits can be used for predicting an NSCLC
prognosis. The kits in these embodiments, can comprise a reagent for
detecting mRNA encoding the amino acid sequence of KIF11 or KOC1, a
reagent for detecting the proteins or reagents for detecting the
biological activity of the KIF11 or KOC1 protein.

[0125] The invention also provides kits for the detection of a compound
that regulates RNA transporting activity. The kits may comprise a cell
expressing a KIF11 polypeptide, or functional equivalent, a KOC1
polypeptide, or functional equivalent, and RNA to be transported, and
DCTN1.

[0126] The kits of the invention may also be used to screen for compounds
for treating or preventing NSCLC. The kits may comprise a KOC1
polypeptide, or functional equivalent, and an RNA that is bound by the
KOC1 polypeptide or functional equivalent. In the present invention, any
RNA transportable with RNA transporter activity of KOC1-KIF11 complex can
be used as the RNA to be transported. Prefer RNA can be selected from
transcripts of genes shown in table 2, or fragment thereof. An RNA to be
transported may also be labeled for detecting RNA transporter activity.
Furthermore, in the present invention, KOC1 and KIF11 polypeptide or
functional equivalent thereof is expressed as fusion protein with signal
generating protein for observation by microscopy or cell imaging systems.
For example, ECFP, EYFP, and EGFP may be used for signal generating
protein.

Array and Pluralities

[0127] The invention also includes a nucleic acid substrate array
comprising one or more of the KIF11, GHSR1b, NTSR1, and FOXM1 genes. The
nucleic acids on the array specifically correspond to one or more
polynucleotide sequences represented by KIF11, GHSR1b, NTSR1, and FOXM1
genes. The expression level of 2, 3 or 4 of the KIF11, GHSR1b, NTSR1, and
FOXM1 genes is identified by detecting the binding of nucleic acid to the
array.

[0128] The invention also includes an isolated plurality (i.e., a mixture
of two or more nucleic acids) of nucleic acids. The nucleic acids are in
a liquid phase or a solid phase, e.g., immobilized on a solid support
such as a nitrocellulose membrane. The plurality includes one or more of
the polynucleotides represented by KIF11, GHSR1b, NTSR1, and FOXM1 genes.
According to a further embodiment of the present invention, the plurality
includes 2, 3, or 4 of the polynucleotides represented by KIF11, GHSR1b,
NTSR1, and FOXM1 genes.

Chips

[0129] The DNA chip is a device that is convenient to compare the
expression levels of a number of genes at the same time. DNA chip-based
expression profiling can be carried out, for example, by the method as
disclosed in "Microarray Biochip Technology" (Mark Schena, Eaton
Publishing, 2000), etc.

[0130] A DNA chip comprises immobilized high-density probes to detect a
number of genes. Thus, the expression levels of many genes can be
estimated at the same time by a single-round analysis. Namely, the
expression profile of a specimen can be determined with a DNA chip. The
DNA chip-based method of the present invention comprises the following
steps of: [0131] (1) synthesizing aRNAs or cDNAs corresponding to the
marker genes; [0132] (2) hybridizing the aRNAs or cDNAs with probes for
marker genes; and [0133] (3) detecting the aRNA or cDNA hybridizing with
the probes and quantifying the amount of mRNA thereof.

[0134] The term "aRNA" refers to RNA transcribed from a template cDNA with
RNA polymerase. An aRNA transcription kit for DNA chip-based expression
profiling is commercially available. With such a kit, aRNA can be
synthesized from T7 promoter-attached cDNA as a template using T7 RNA
polymerase. On the other hand, by PCR using random primer, cDNA can be
amplified using as a template a cDNA synthesized from mRNA.

[0135] Alternatively, the DNA chip comprises probes, which have been
spotted thereon, to detect the marker genes of the present invention
(KIF11, GHSR1b, NTSR1 or FOXM1 gene). There is no limitation on the
number of marker genes spotted on the DNA chip, and 1, 2, 3 or all of the
genes, KIF11, GHSR1b, NTSR1, and FOXM1, may be used. Any other genes as
well as the marker genes can be spotted on the DNA chip. For example, a
probe for a gene whose expression level is hardly altered may be spotted
on the DNA chip. Such a gene can be used to normalize assay results when
the assay results are intended to be compared between multiple chips or
between different assays.

[0136] A probe is designed for each marker gene selected, and spotted on a
DNA chip. Such a probe may be, for example, an oligonucleotide comprising
5-50 nucleotide residues. A method for synthesizing such oligonucleotides
on a DNA chip is known to those skilled in the art. Longer DNAs can be
synthesized by PCR or chemically. A method for spotting long DNA, which
is synthesized by PCR or the like, onto a glass slide is also known to
those skilled in the art. A DNA chip that is obtained by the method as
described above can be used for diagnosing NSCLC according to the present
invention.

[0137] The prepared DNA chip is contacted with aRNA, followed by the
detection of hybridization between the probe and aRNA. The aRNA can be
previously labeled with a fluorescent dye. A fluorescent dye such as Cy3
(red) and Cy5 (green) can be used to label an aRNA. aRNAs from a subject
and a control are labeled with different fluorescent dyes, respectively.
The difference in the expression level between the two can be estimated
based on a difference in the signal intensity. The signal of fluorescent
dye on the DNA chip can be detected by a scanner and analyzed using a
special program. For example, the Suite from Affymetrix is a software
package for DNA chip analysis.

Identifying Compounds that Inhibit NSCLC-Associated Gene Expression

[0138] A compound that inhibits the expression or activity of a target
NSCLC-associated gene (KIF11, GHSR1b, NTSR1 or FOXM1 gene) is identified
by contacting a test cell expressing the NSCLC-associated gene with a
test compound and determining the expression level or activity of the
NSCLC-associated gene. A decrease in expression compared to the normal
control level indicates that the compound is an inhibitor of the
NSCLC-associated gene. Such compounds identified according to the method
are useful for inhibiting NSCLC.

[0139] The test cell may be a population of cells and includes any cells
as long as the cell expresses the target NSCLC-associated gene(s). For
example, the test cell may be an immortalized cell line derived from an
NSCLC cell. Alternatively, the test cell may be a cell transfected with
any of the KIF11, GHSR1b, NTSR1, and FOXM1 genes, or which has been
transfected with the regulatory sequence (e.g., promoter) of any of the
genes that is operably linked to a reporter gene.

Screening Compounds

[0140] Using KIF11, GHSR1b, NTSR1 or FOXM1 gene, proteins encoded by the
gene or transcriptional regulatory region of the gene, compounds can be
screened that alter the expression of the gene or biological activity of
a polypeptide encoded by the gene. Such compounds are expected to serve
as pharmaceuticals for treating or preventing NSCLC.

[0141] Therefore, the present invention provides a method of screening for
a compound for treating or preventing NSCLC using the polypeptide of the
present invention. An embodiment of this screening method comprises the
steps of: (a) contacting a test compound with a polypeptide encoded by
KIF11, GHSR1b, NTSR1 or FOXM1 gene; (b) detecting the binding activity
between the polypeptide of the present invention and the test compound;
and (c) selecting the compound that binds to the polypeptide.

[0142] As explained in more detail below, KOC1 and KIF11 form a complex
that has RNA transporting activity. Thus, the present invention also
provides methods of identifying polypeptides and other compounds that
modulate RNA transport activity. For example, a polypeptide can be tested
for RNA transporting activity by contacting a KIF11 polypeptide (SEQ ID
NO: 2) or a functional equivalent thereof with an RNA that can be
transported by KIF11 under conditions suitable for transportation of RNA.
The level of RNA transported can be measured using well known techniques,
such as by RNA immunoprecipitation, as described in detail below.

[0143] A functional equivalent of a KIF11 polypeptide is a polypeptide
that has a biological activity equivalent to a polypeptide consisting of
the amino acid sequence of SEQ ID NO: 2 and, for example, comprising the
amino acid sequence of SEQ ID NO: 2 (KIF11), wherein one or more amino
acids (usually less than five) are substituted, deleted, or inserted.
Alternatively, the polypeptide may be one that comprises an amino acid
sequence having at least about 80% homology (also referred to as sequence
identity) to SEQ ID NO: 2. In other embodiments, the polypeptide can be
encoded by a polynucleotide that hybridizes under stringent conditions
(as defined below) to a polynucleotide consisting of the nucleotide
sequence of SEQ ID NO: 1.

[0144] In some embodiments, the KIF11 polypeptide or functional equivalent
is contacted with a KOC1 polypeptide or functional equivalent thereof. A
functional equivalent of a KOC1 polypeptide is a polypeptide that has a
biological activity equivalent to a polypeptide consisting of the amino
acid sequence of SEQ ID NO: 105 and, for example, comprising the amino
acid sequence of SEQ ID NO: 105, wherein one or more amino acids (usually
less than five) are substituted, deleted, or inserted. Alternatively, the
polypeptide may be one that comprises an amino acid sequence having at
least about 80% homology (also referred to as sequence identity) to SEQ
ID NO: 105. In other embodiments, the polypeptide can be encoded by a
polynucleotide that hybridizes under stringent conditions (as defined
below) to a polynucleotide consisting of the nucleotide sequence of SEQ
ID NO: 104. In some embodiments, a functional equivalent comprises at
least one RRM or KH domain.

[0145] The invention also provides methods of identifying agents that
modulate RNA transporting activity. In these methods, an agent suspected
of modulating RNA transporting activity with a KIF11 polypeptide or
functional equivalent. The level of transported RNA is detected and
compared to the level in a control in the absence of the agent.

[0146] The polypeptide to be used for the screening may be a recombinant
polypeptide or a protein derived from the nature or a partial peptide
thereof. The polypeptide to be contacted with a test compound can be, for
example, a purified polypeptide, a soluble protein, a form bound to a
carrier or a fusion protein fused with other polypeptides.

[0147] As a method of screening for proteins that bind to KIF11, GHSR1b,
NTSR1 or FOXM1 polypeptide, many methods well known by a person skilled
in the art can be used. Such a screening can be conducted by, for
example, immunoprecipitation method using methods well known in the art.
The proteins of the invention can be recombinantly produced using
standard procedures. For example, a gene encoding any of the KIF11,
GHSR1b, NTSR1, and FOXM1 polypeptides is expressed in animal cells by
inserting the gene into an expression vector for foreign genes, such as
pSV2neo, pcDNA I, pcDNA3.1, pCAGGS and pCD8. The promoter to be used for
the expression may be any promoter that can be used commonly and include,
for example, the SV40 early promoter (Rigby in Williamson (ed.), Genetic
Engineering, vol. 3. Academic Press, London, 83-141 (1982)), the
EF-α promoter (Kim et al., Gene 91: 217-23 (1990)), the CAG
promoter (Niwa et al., Gene 108: 193-200 (1991)), the RSV LTR promoter
(Cullen, Methods in Enzymology 152: 684-704 (1987)) the SRα
promoter (Takebe et al., Mol Cell Biol 8: 466 (1988)), the CMV immediate
early promoter (Seed and Aruffo, Proc Natl Acad Sci USA 84: 3365-9
(1987)), the SV40 late promoter (Gheysen and Fiers, J Mol Appl Genet 1:
385-94 (1982)), the Adenovirus late promoter (Kaufman et al., Mol Cell
Biol 9: 946 (1989)), the HSV TK promoter and so on. The introduction of
the gene into animal cells to express a foreign gene can be performed
according to any methods, for example, the electroporation method (Chu et
al., Nucleic Acids Res 15: 1311-26 (1987)), the calcium phosphate method
(Chen and Okayama, Mol Cell Biol 7: 2745-52 (1987)), the DEAE dextran
method (Lopata et al., Nucleic Acids Res 12: 5707-17 (1984); Sussman and
Milman, Mol Cell Biol 4: 1642-3 (1985)), the Lipofectin method (Derijard,
B Cell 7: 1025-37 (1994); Lamb et al., Nature Genetics 5: 22-30 (1993):
Rabindran et al., Science 259: 230-4 (1993)), and so on. The NSC
polypeptide can also be expressed as a fusion protein comprising a
recognition site (epitope) of a monoclonal antibody by introducing the
epitope of the monoclonal antibody, whose specificity has been revealed,
to the N- or C-terminus of the polypeptide. A commercially available
epitope-antibody system can be used (Experimental Medicine 13: 85-90
(1995)). Vectors which can express a fusion protein with, for example,
β-galactosidase, maltose binding protein, glutathione S-transferase,
green florescence protein (GFP), and so on, by the use of its multiple
cloning sites are commercially available.

[0148] A fusion protein prepared by introducing only small epitopes
consisting of several to a dozen amino acids so as not to change the
property of the original polypeptide by the fusion is also reported.
Epitopes, such as polyhistidine (His-tag), influenza aggregate HA, human
c-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10
protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag
(an epitope on monoclonal phage) and such, and monoclonal antibodies
recognizing them can be used as the epitope-antibody system for screening
proteins binding to the KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide
(Experimental Medicine 13: 85-90 (1995)).

[0149] In immunoprecipitation, an immune complex is formed by adding these
antibodies to cell lysate prepared using an appropriate detergent. The
immune complex consists of the KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide,
a polypeptide comprising the binding ability with the polypeptide, and an
antibody. Immunoprecipitation can be also conducted using antibodies
against the KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide, in addition to the
use of antibodies against the above epitopes, which antibodies can be
prepared according to conventional methods and may be in any form, such
as monoclonal or polyclonal antibodies, and includes antiserum obtained
by immunizing an animal such as a rabbit with the polypeptide, all
classes of polyclonal and monoclonal antibodies, as well as recombinant
antibodies (e.g., humanized antibodies).

[0150] Specifically, antibodies against KIF11, GHSR1b, NTSR1 or FOXM1
polypeptide can be prepared using techniques well known in the art. For
example, KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide used as an antigen to
obtain an antibody may be derived from any animal species, but preferably
is derived from a mammal such as a human, mouse, or rat, more preferably
from a human. The polypeptide used as the antigen can be recombinantly
produced or isolated from natural sources. According to the present
invention, the polypeptide to be used as an immunization antigen may be a
complete protein or a partial peptide of the KIF11, GHSR1b, NTSR1 or
FOXM1 polypeptide. A partial peptide may comprise, for example, the amino
(N)-terminal or carboxy (C)-terminal fragment of the KIF11, GHSR1b, NTSR1
or FOXM1 polypeptide.

[0151] Any mammalian animal may be immunized with the antigen, but
preferably the compatibility with parental cells used for cell fusion is
taken into account. In general, animals of Rodentia, Lagomorpha or
Primates are used. Animals of Rodentia include, for example, mouse, rat
and hamster. Animals of Lagomorpha include, for example, rabbit. Animals
of Primates include, for example, a monkey of Catarrhini (old world
monkey) such as Macaca fascicularis, rhesus monkey, sacred baboon and
chimpanzees.

[0152] Methods for immunizing animals with antigens are known in the art.
Intraperitoneal injection or subcutaneous injection of antigens is a
standard method for immunization of mammals. More specifically, antigens
may be diluted and suspended in an appropriate amount of phosphate
buffered saline (PBS), physiological saline, etc. If desired, the antigen
suspension may be mixed with an appropriate amount of a standard
adjuvant, such as Freund's complete adjuvant, made into emulsion, and
then administered to mammalian animals. Preferably, it is followed by
several administrations of the antigen mixed with an appropriately amount
of Freund's incomplete adjuvant every 4 to 21 days. An appropriate
carrier may also be used for immunization. After immunization as above,
the serum is examined by a standard method for an increase in the amount
of desired antibodies.

[0153] Polyclonal antibodies against KIF11, GHSR1b, NTSR1 or FOXM1
polypeptide may be prepared by collecting blood from the immunized mammal
examined for the increase of desired antibodies in the serum, and by
separating serum from the blood by any conventional method. Polyclonal
antibodies include serum containing the polyclonal antibodies, as well as
the fraction containing the polyclonal antibodies may be isolated from
the serum. Immunoglobulin G or M can be prepared from a fraction which
recognizes only the KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide using, for
example, an affinity column coupled with the polypeptide, and further
purifying this fraction using protein A or protein G column.

[0154] To prepare monoclonal antibodies, immune cells are collected from
the mammal immunized with the antigen and checked for the increased level
of desired antibodies in the serum as described above, and are subjected
to cell fusion. The immune cells used for cell fusion are preferably
obtained from spleen. Other preferred parental cells to be fused with the
above immunocyte include, for example, myeloma cells of mammalians, and
more preferably myeloma cells having an acquired property for the
selection of fused cells by drugs.

[0155] The above immunocyte and myeloma cells can be fused according to
known methods, for example, the method of Milstein et al., (Galfre and
Milstein, Methods Enzymol 73: 3-46 (1981)).

[0156] Resulting hybridomas obtained by the cell fusion may be selected by
cultivating them in a standard selection medium, such as HAT medium
(hypoxanthine, aminopterin, and thymidine containing medium). The cell
culture is typically continued in the HAT medium for several days to
several weeks, the time being sufficient to allow all the other cells,
with the exception of the desired hybridoma (non-fused cells), to die.
Then, the standard limiting dilution is performed to screen and clone a
hybridoma cell producing the desired antibody.

[0157] In addition to the above method, in which a non-human animal is
immunized with an antigen for preparing hybridoma, human lymphocytes such
as those infected by EB virus may be immunized with KIF11, GHSR1b, NTSR1
or FOXM1 polypeptide, cells expressing the polypeptide, or their lysates
in vitro. Then, the immunized lymphocytes are fused with human-derived
myeloma cells that are capable of indefinitely dividing, such as U266, to
yield a hybridoma producing a desired human antibody that is able to bind
to the KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide can be obtained
(Unexamined Published Japanese Patent Application No. (JP-A) Sho
63-17688).

[0158] The obtained hybridomas are subsequently transplanted into the
abdominal cavity of a mouse and the ascites are extracted. The obtained
monoclonal antibodies can be purified by, for example, ammonium sulfate
precipitation, a protein A or protein G column, DEAE ion exchange
chromatography, or an affinity column to which any of the target proteins
of the present invention (KIF11, GHSR1b, NTSR1, and FOXM1 polypeptide) is
coupled. The antibody can be used not only in the present screening
method, but also for purification and detection of KIF11, GHSR1b, NTSR1
or FOXM1 polypeptide, and serve also as candidates for agonists and
antagonists of the polypeptide. In addition, this antibody can be applied
to the antibody treatment for diseases related to the KIF11, GHSR1b,
NTSR1 or FOXM1 polypeptide including NSCLC as described infra.

[0159] Monoclonal antibodies thus obtained can be also recombinantly
prepared using genetic engineering techniques (see, for example,
Borrebaeck and Larrick, Therapeutic Monoclonal Antibodies, published in
the United Kingdom by MacMillan Publishers LTD (1990)). For example, a
DNA encoding an antibody may be cloned from an immune cell, such as a
hybridoma or an immunized lymphocyte producing the antibody, inserted
into an appropriate vector, and introduced into host cells to prepare a
recombinant antibody. Such recombinant antibody can also be used for the
present screening.

[0160] Furthermore, an antibody used in the screening and so on may be a
fragment of an antibody or modified antibody, so long as it binds to one
or more of KIF11, GHSR1b, NTSR1, and FOXM1 polypeptides. For instance,
the antibody fragment may be Fab, F(ab')2, Fv, or single chain Fv
(scFv), in which Fv fragments from H and L chains are ligated by an
appropriate linker (Huston et al., Proc Natl Acad Sci USA 85: 5879-83
(1988)). More specifically, an antibody fragment may be generated by
treating an antibody with an enzyme, such as papain or pepsin.
Alternatively, a gene encoding the antibody fragment may be constructed,
inserted into an expression vector, and expressed in an appropriate host
cell (see, for example, Co et al., J Immunol 152: 2968-76 (1994); Better
and Horwitz, Methods Enzymol 178: 476-96 (1989); Pluckthun and Skerra,
Methods Enzymol 178: 497-515 (1989); Lamoyi, Methods Enzymol 121: 652-63
(1986); Rousseaux et al., Methods Enzymol 121: 663-9 (1986); Bird and
Walker, Trends Biotechnol 9: 132-7 (1991)).

[0161] An antibody may be modified by conjugation with a variety of
molecules, such as polyethylene glycol (PEG). Modified antibodies can be
obtained through chemically modification of an antibody. These
modification methods are conventional in the field.

[0162] Alternatively, an antibody may be obtained as a chimeric antibody,
between a variable region derived from nonhuman antibody and the constant
region derived from human antibody, or as a humanized antibody,
comprising the complementarity determining region (CDR) derived from
nonhuman antibody, the frame work region (FR) derived from human
antibody, and the constant region. Such antibodies can be prepared using
known technology.

[0163] Humanization can be performed by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody (see e.g.,
Verhoeyen et al., Science 239:1534-1536 (1988)). Accordingly, such
humanized antibodies are chimeric antibodies, wherein substantially less
than an intact human variable domain has been substituted by the
corresponding sequence from a non-human species.

[0164] Fully human antibodies comprising human variable regions in
addition to human framework and constant regions can also be used. Such
antibodies can be produced using various techniques known in the art. For
example in vitro methods involve use of recombinant libraries of human
antibody fragments displayed on bacteriophage (e.g., Hoogenboom & Winter,
J. Mol. Biol. 227:381 (1991), Similarly, human antibodies can be made by
introducing of human immunoglobulin loci into transgenic animals, e.g.,
mice in which the endogenous immunoglobulin genes have been partially or
completely inactivated. This approach is described, e.g., in U.S. Pat.
Nos. 6,150,584, 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;
5,661,016.

[0165] Antibodies obtained as above may be purified to homogeneity. For
example, the separation and purification of the antibody can be performed
according to separation and purification methods used for general
proteins. For example, the antibody may be separated and isolated by the
appropriately selected and combined use of column chromatographies, such
as affinity chromatography, filter, ultrafiltration, salting-out,
dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing,
and others (Antibodies: A Laboratory Manual. Ed Harlow and David Lane,
Cold Spring Harbor Laboratory (1988)), but are not limited thereto. A
protein A column and protein G column can be used as the affinity column.
Exemplary protein A columns to be used include, for example, Hyper D,
POROS, and Sepharose F.F. (Pharmacia).

[0167] An immune complex can be precipitated, for example with Protein A
sepharose or Protein G sepharose when the antibody is a mouse IgG
antibody. If the KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide is prepared as
a fusion protein with an epitope, such as GST, an immune complex can be
formed in the same manner as in the use of the antibody against the
KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide, using a substance specifically
binding to these epitopes, such as glutathione-Sepharose 4B.

[0168] Immunoprecipitation can be performed by following or according to,
for example, the methods in the literature (Harlow and Lane, Antibodies,
511-52, Cold Spring Harbor Laboratory publications, New York (1988)).

[0169] SDS-PAGE is commonly used for analysis of immunoprecipitated
proteins and the bound protein can be analyzed by the molecular weight of
the protein using gels with an appropriate concentration. Since the
protein bound to the KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide is
difficult to detect by a common staining method, such as Coomassie
staining or silver staining, the detection sensitivity for the protein
can be improved by culturing cells in culture medium containing
radioactive isotope, 35S-methionine or 35S-cystein, labeling
proteins in the cells, and detecting the proteins. The target protein can
be purified directly from the SDS-polyacrylamide gel and its sequence can
be determined, when the molecular weight of the protein has been
revealed.

[0170] As a method for screening proteins binding to any of KIF11, GHSR1b,
NTSR1, and FOXM1 polypeptides using the polypeptide, for example,
West-Western blotting analysis (Skolnik et al., Cell 65: 83-90 (1991))
can be used. Specifically, a protein binding to KIF11, GHSR1b, NTSR1 or
FOXM1 polypeptide can be obtained by preparing a cDNA library from cells,
tissues, organs (for example, tissues such as lung cells) or cultured
cells (particularly those derived from NSCLC cells) expected to express a
protein binding to the KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide using a
phage vector (e.g., ZAP), expressing the protein on LB-agarose, fixing
the protein expressed on a filter, reacting the purified and labeled
KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide with the above filter, and
detecting the plaques expressing proteins bound to the KIF11, GHSR1b,
NTSR1 or FOXM1 polypeptide according to the label. The KIF11, GHSR1b,
NTSR1 or FOXM1 polypeptide may be labeled by utilizing the binding
between biotin and avidin, or by utilizing an antibody that specifically
binds to the KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide, or a peptide or
polypeptide (for example, GST) that is fused to the KIF11, GHSR1b, NTSR1
or FOXM1 polypeptide. Methods using radioisotope or fluorescence and such
may be also used.

[0172] In the two-hybrid system, KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide
is fused to the SRF-binding region or GAL4-binding region and expressed
in yeast cells. A cDNA library is prepared from cells expected to express
a protein binding to the KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide, such
that the library, when expressed, is fused to the VP16 or GAL4
transcriptional activation region. The cDNA library is then introduced
into the above yeast cells and the cDNA derived from the library is
isolated from the positive clones detected (when a protein binding to the
polypeptide of the invention is expressed in yeast cells, the binding of
the two activates a reporter gene, making positive clones detectable). A
protein encoded by the cDNA can be prepared by introducing the cDNA
isolated above to E. coli and expressing the protein.

[0173] As a reporter gene, for example, Ade2 gene, lacZ gene, CAT gene,
luciferase gene and such can be used in addition to the HIS3 gene.

[0174] A compound binding to KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide can
also be screened using affinity chromatography. For example, KIF11,
GHSR1b, NTSR1 or FOXM1 polypeptide may be immobilized on a carrier of an
affinity column, and a test compound, containing a protein capable of
binding to KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide, is applied to the
column. A test compound herein may be, for example, cell extracts, cell
lysates, etc. After loading the test compound, the column is washed, and
compounds bound to KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide can be
prepared.

[0175] When the test compound is a protein, the amino acid sequence of the
obtained protein is analyzed, an oligo DNA is synthesized based on the
sequence, and cDNA libraries are screened using the oligo DNA as a probe
to obtain a DNA encoding the protein.

[0176] A biosensor using the surface plasmon resonance phenomenon may be
used as a mean for detecting or quantifying the bound compound in the
present invention. When such a biosensor is used, the interaction between
KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide and a test compound can be
observed real-time as a surface plasmon resonance signal, using only a
minute amount of polypeptide and without labeling (for example, BIAcore,
Pharmacia). Therefore, it is possible to evaluate the binding between
KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide and a test compound using a
biosensor such as BIAcore.

[0177] The methods of screening for molecules that bind when an
immobilized KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide is exposed to
synthetic chemical compounds, or natural substance banks or a random
phage peptide display library, and the methods of screening using
high-throughput based on combinatorial chemistry techniques (Wrighton et
al., Science 273: 458-64 (1996); Verdine, Nature 384: 11-13 (1996);
Hogan, Nature 384: 17-9 (1996)) to isolate not only proteins but chemical
compounds that bind to KIF11, GHSR1b, NTSR1 or FOXM1 protein (including
agonist and antagonist) are well known to one skilled in the art.

[0178] Alternatively, the present invention provides a method of screening
for a compound for treating or preventing NSCLC using KIF11, GHSR1b,
NTSR1 or FOXM1 polypeptide comprising the steps as follows: [0179] (a)
contacting a test compound with KIF11, GHSR1b, NTSR1 or FOXM1
polypeptide; [0180] (b) detecting the biological activity of the KIF11,
GHSR1b, NTSR1 or FOXM1 polypeptide of step (a); and [0181] (c) selecting
a compound that suppresses the biological activity of the KIF11, GHSR1b,
NTSR1 or FOXM1 polypeptide in comparison with the biological activity
detected in the absence of the test compound.

[0182] Since proteins encoded by any of the genes of KIF11, GHSR1b, NTSR1,
and FOXM1 have the activity of promoting cell proliferation of NSCLC
cells, a compound which inhibits this activity of one of these proteins
can be screened using this activity as an index.

[0183] Any polypeptides can be used for screening so long as they comprise
the biological activity of KIF11, GHSR1b, NTSR1 or FOXM1 proteins. Such
biological activity includes cell-proliferating activity and binding
ability to other proteins of the proteins encoded by KIF11, GHSR1b, NTSR1
or FOXM1 gene. For example, a human protein encoded by KIF11, GHSR1b,
NTSR1 or FOXM1 gene can be used and polypeptides functionally equivalent
to these proteins can also be used. Such polypeptides may be expressed
endogenously or exogenously by cells.

[0184] The compound isolated by this screening is a candidate for
antagonists of the KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide. The term
"antagonist" refers to molecules that inhibit the function of KIF11,
GHSR1b, NTSR1 or FOXM1 polypeptide by binding thereto. Moreover, a
compound isolated by this screening is a candidate for compounds which
inhibit the in vivo interaction of KIF11, GHSR1b, NTSR1 or FOXM1
polypeptide with molecules (including DNAs and proteins).

[0185] When the biological activity to be detected in the present method
is cell proliferation, it can be detected, for example, by preparing
cells which express KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide, culturing
the cells in the presence of a test compound, and determining the speed
of cell proliferation, measuring the cell cycle and such, as well as by
measuring the colony forming activity.

[0186] As discussed in detail above, by controlling the expression levels
of KIF11, GHSR1b, NTSR1 or FOXM1 gene, one can control the onset and
progression of NSCLC. Thus, compounds that may be used in the treatment
or prevention of NSCLC, can be identified through screenings that use the
expression levels of one or more of KIF11, GHSR1b, NTSR1, and FOXM1 genes
as indices. In the context of the present invention, such screening may
comprise, for example, the following steps: [0187] (a) contacting a
test compound with a cell expressing one or more of KIF11, GHSR1b, NTSR1,
and FOXM1 genes; and [0188] (b) selecting a compound that reduces the
expression level of one or more of the genes in comparison with the
expression level detected in the absence of the test compound.

[0189] Cells expressing at least one of KIF11, GHSR1b, NTSR1, and FOXM1
genes include, for example, cell lines established from NSCLC cells; such
cells can be used for the above screening of the present invention (e.g.,
A549, NCI-H226, NCI-H522, LC319). The expression level can be estimated
by methods well known to one skilled in the art. In the method of
screening, a compound that reduces the expression level of at least one
of the genes can be selected as candidate agents to be used for the
treatment or prevention of NSCLC.

[0190] Alternatively, the screening method of the present invention may
comprise the following steps: [0191] (a) contacting a test compound
with a cell into which a vector comprising the transcriptional regulatory
region of one or more of the marker genes and a reporter gene that is
expressed under the control of the transcriptional regulatory region has
been introduced, wherein the marker genes are selected from the group of
KIF11, GHSR1b, NTSR1, and FOXM1; [0192] (b) measuring the activity of
said reporter gene; and [0193] (c) selecting a compound that reduces the
expression level of said reporter gene as compared to a control.

[0194] Suitable reporter genes and host cells are well known in the art.
The reporter construct required for the screening can be prepared using
the transcriptional regulatory region of a marker gene. When the
transcriptional regulatory region of a marker gene has been known to
those skilled in the art, a reporter construct can be prepared using the
previous sequence information. When the transcriptional regulatory region
of a marker gene remains unidentified, a nucleotide segment containing
the transcriptional regulatory region can be isolated from a genome
library based on the nucleotide sequence information of the marker gene
(e.g., based the 5' upstream sequence information).

[0195] In a further embodiment of the method of screening for a compound
for treating or preventing NSCLC of the present invention, the method
utilizes the binding ability of KIF11 to KOC1, or GHSR1b or NTSR1 to NMU.

[0196] As described above, the present inventors revealed that KOC1 not
only co-localized with KIF11 in human normal tissues, NSCLCs, and cell
lines, but also directly interacted with KIF11 in NSCLC cells in vitro,
and that the treatment of NSCLC cells with siRNAs for KIF11 reduced its
expression and led to growth suppression. The results suggest that
KOC1-KIF11 signaling affects growth of NSCLC cells. Thus, it is expected
that the inhibition of the binding between KOC1 and KIF11 leads to the
suppression of cell proliferation, and compounds inhibiting the binding
serve as pharmaceuticals for treating or preventing NSCLCs. This
screening method includes the steps of: (a) contacting a KIF11
polypeptide or functional equivalent thereof with KOC1, or a functional
equivalent thereof, in the presence of a test compound; (b) detecting the
binding between the polypeptide and KOC1; and (c) selecting the test
compound that inhibits the binding between the polypeptide and KOC1.

[0197] Furthermore, as described above, the present inventors revealed
GHSR1b and NTSR1 as the likely targets for the growth-promoting effect of
NMU in lung tumors. The present inventors revealed that NMU-25 bound to
these receptors on the cell surface, and that treatment of NSCLC cells
with siRNAs for GHSR1 or NTSR1 reduced expression of the receptors and
led to apoptosis. The results suggest that NMU affects growth of NSCLC
cells by acting through GHSR1b and/or NTSR1 (FIG. 14). Thus, it is
expected that the inhibition of binding between GHSR1b or NTSR1 and NMU
leads to the suppression of cell proliferation, and compounds inhibiting
the binding serve as pharmaceuticals for treating or preventing NSCLCs.
This screening method includes the steps of: (a) contacting a GHSR1b or
NTSR1 polypeptide or functional equivalent thereof with NMU in the
presence of a test compound; (b) detecting binding between the
polypeptide and NMU; and (c) selecting the test compound that inhibits
binding between the polypeptide and NMU.

[0198] KOC1 and KIF11 polypeptides, or GHSR1b or NTSR1 and NMU
polypeptides to be used for the screening may be a recombinant
polypeptide or a protein derived from the nature, or may also be a
partial peptide thereof so long as it retains the binding ability to each
other. Such partial peptides retaining the binding ability are herein
referred to as "functional equivalents". The KOC1 and KIF11 polypeptides,
or GHSR1b or NTSR1 and NMU polypeptides to be used in the screening can
be, for example, a purified polypeptide, a soluble protein, a form bound
to a carrier or a fusion protein fused with other polypeptides.

[0199] As a method of screening for compounds that inhibit binding between
KOC1 and KIF11, or GHSR1b or NTSR1 and NMU, many methods well known by
one skilled in the art can be used. Such a screening can be carried out
as an in vitro assay system, for example, in a cellular system. More
specifically, first, either KOC1 or KIF11, or GHSR1b or NTSR1, or NMU is
bound to a support, and the other protein is added together with a test
compound thereto. Next, the mixture is incubated, washed and the other
protein bound to the support is detected and/or measured.

[0200] Examples of supports that may be used for binding proteins include
insoluble polysaccharides, such as agarose, cellulose and dextran; and
synthetic resins, such as polyacrylamide, polystyrene and silicon;
preferably commercial available beads and plates (e.g., multi-well
plates, biosensor chip, etc.) prepared from the above materials may be
used. When using beads, they may be filled into a column. Alternatively,
the use of magnetic beads of also known in the art, and enables to
readily isolate proteins bound on the beads via magnetism.

[0201] The binding of a protein to a support may be conducted according to
routine methods, such as chemical bonding and physical adsorption.
Alternatively, a protein may be bound to a support via antibodies
specifically recognizing the protein. Moreover, binding of a protein to a
support can be also conducted by means of avidin and biotin.

[0202] The binding between proteins is carried out in buffer, for example,
but are not limited to, phosphate buffer and Tris buffer, as long as the
buffer does not inhibit binding between the proteins.

[0203] In the present invention, a biosensor using the surface plasmon
resonance phenomenon may be used as a mean for detecting or quantifying
the bound protein. When such a biosensor is used, the interaction between
the proteins can be observed real-time as a surface plasmon resonance
signal, using only a minute amount of polypeptide and without labeling
(for example, BIAcore, Pharmacia). Therefore, it is possible to evaluate
binding between the KOC1 and KIF11, or GHSR1b or NTSR1 and NMU using a
biosensor such as BIAcore.

[0204] Alternatively, either KOC1 or KIF11, or GHSR1b or NTSR1, or NMU may
be labeled, and the label of the bound protein may be used to detect or
measure the bound protein. Specifically, after pre-labeling one of the
proteins, the labeled protein is contacted with the other protein in the
presence of a test compound, and then bound proteins are detected or
measured according to the label after washing.

[0205] Labeling substances such as radioisotope (e.g., 3H, 14C,
32P, 33P, 35S, 125I, 131I), enzymes (e.g.,
alkaline phosphatase, horseradish peroxidase, β-galactosidase,
β-glucosidase), fluorescent substances (e.g., fluorescein
isothiosyanete (FITC), rhodamine) and biotin/avidin, may be used for the
labeling of a protein in the present method. When the protein is labeled
with radioisotope, the detection or measurement can be carried out by
liquid scintillation. Alternatively, proteins labeled with enzymes can be
detected or measured by adding a substrate of the enzyme to detect the
enzymatic change of the substrate, such as generation of color, with
absorptiometer. Further, in case where a fluorescent substance is used as
the label, the bound protein may be detected or measured using
fluorophotometer.

[0206] Furthermore, binding of KOC1 and KIF11, or GHSR1b or NTSR1 and NMU
can be also detected or measured using antibodies to the KOC1 and KIF11,
or GHSR1b or NTSR1 and NMU. For example, after contacting the KOC1
polypeptide immobilized on a support with a test compound and KIF11, the
mixture is incubated and washed, and detection or measurement can be
conducted using an antibody against KIF11. Alternatively, KIF11 may be
immobilized on a support, and an antibody against KOC1 may be used as the
antibody. When the combination of GHSR1b or NTSR1 and NMU is used, GHSR1b
or NTSR1 polypeptide may be immobilized on a support with a test compound
and NMU, the mixture is incubated and washed, and detection or
measurement can be conducted using an antibody against NMU.
Alternatively, NMU may be immobilized on a support, and an antibody
against GHSR1b or NTSR1 may be used as the antibody.

[0207] In case of using an antibody in the present screening, the antibody
is preferably labeled with one of the labeling substances mentioned
above, and detected or measured based on the labeling substance.
Alternatively, the antibody against KOC1 or KIF11, or GHSR1b or NTSR1, or
NMU may be used as a primary antibody to be detected with a secondary
antibody that is labeled with a labeling substance. Furthermore, the
antibody bound to the protein in the screening of the present invention
may be detected or measured using protein G or protein A column.

[0209] In the two-hybrid system, for example, KOC1 polypeptide is fused to
the SRF-binding region or GAL4-binding region and expressed in yeast
cells. KIF11 polypeptide that binds to KOC1 polypeptide is fused to the
VP16 or GAL4 transcriptional activation region and also expressed in the
yeast cells in the existence of a test compound. Alternatively, KIF11
polypeptide may be fused to the SRF-binding region or GAL4-binding
region, and KOC1 polypeptide to the VP16 or GAL4 transcriptional
activation region. When the combination of GHSR1b or NTSR1 and NMU is
used in the two-hybrid system, for example, GHSR1b or NTSR1 polypeptide
is fused to the SRF-binding region or GAL4-binding region and expressed
in yeast cells. NMU polypeptide that binds to GHSR1b or NTSR1 polypeptide
is fused to the VP16 or GAL4 transcriptional activation region and also
expressed in the yeast cells in the existence of a test compound.
Alternatively, NMU polypeptide may be fused to the SRF-binding region or
GAL4-binding region, and GHSR1b or NTSR1 polypeptide to the VP16 or GAL4
transcriptional activation region. When the test compound does not
inhibit the binding between KOC1 and KIF11, or GHSR1b or NTSR1 and NMU,
the binding of the two activates a reporter gene, making positive clones
detectable.

[0210] As a reporter gene, for example, Ade2 gene, lacZ gene, CAT gene,
luciferase gene and such can be used besides HIS3 gene.

[0211] Moreover, when the combination of GHSR1b or NTSR1 and NMU is used
in the screening method, since GHSR1b and NTSR1 are polypeptides
naturally expressed on the cell surface, in a preferable embodiment of
the present screening method, the polypeptides are expressed on the
surface of a living cell. When the polypeptides are expressed on the
surface of a living cell, the binding between the polypeptide and NMU can
be detected by methods detecting the autocrine and paracrine signaling
leading to stimulation of tumor cell growth (Heasley, Oncogene 20:
1563-1569 (2001)). For example, the binding between GHSR1 or NTSR1
polypeptide and NMU can be detected by: [0212] (1) detecting the
concentration of calcium or cAMP in the cell (e.g. FLIPR assay, Biochem.
Biophys. Res. Commun. 276: 435-438, 2000; Nature 406: 70-74, 2000; J.
Biol. Chem. 275:21068-21074, 2000); [0213] (2) detecting the activation
of the polypeptide; [0214] (3) detecting the interaction between the
polypeptide and G-protein (e.g. FLIPR assay, Biochem. Biophys. Res.
Commun. 276: 435-438, 2000; Nature 406: 70-74, 2000; J. Biol. Chem.
275:21068-21074, 2000, or binding assay with 125I labeled peptide);
[0215] (4) detecting the activation of phospholipase C or its down stream
pathway (Oncogene 20:1563-1569, 2001); [0216] (5) detecting the
activation of kinases of the protein kinase cascade, such as Raf, MEK,
ERKs, and protein kinase D (PKD) (Oncogene 20:1563-1569, 2001); [0217]
(6) detecting the activation of a member of Src/Tec/Bmx-family of
tyrosine kinases (Oncogene 20:1563-1569, 2001); [0218] (7) detecting the
activation of a member of the Ras and Rho family, regulation of a member
of the JNK members of MAP families, or the reorganization of the actin
cytoskeleton (Oncogene 20:1563-1569, 2001); [0219] (8) detecting the
activation of any signal complex mediated by the polypeptide activation;
[0220] (9) detecting the change in subcellular localization of the
polypeptide including the ligand-induced internalization/endocytosis of
the polypeptide (J. Cell Sci., 113: 2963-2975, 2000; J. Histochem.
Cytochem. 48:1553-1563, 2000; Endocrinology Oct. 23, 2003. as doi: 10.
1210/en. 2003-0974); [0221] (10) detecting the activation of any
transcription factor downstream of the polypeptides or the activation of
their downstream gene; and [0222] (11) detecting cell proliferation,
transformation, or any other oncogenic phenotype of the cell.

[0224] A compound isolated by the screening methods of the present
invention is a candidate for drugs which inhibit the activity of KIF11,
GHSR1b, NTSR1 or FOXM1 polypeptide, for treating or preventing diseases
attributed to, for example, cell proliferative diseases, such as NSCLC. A
compound in which a part of the structure of the compound obtained by the
present screening methods of the present invention is converted by
addition, deletion and/or replacement, is included in the compounds
obtained by the screening methods of the present invention. A compound
effective in suppressing the expression of over-expressed genes, i.e.,
KIF11, GHSR1b, NTSR1 or FOXM1 gene, is deemed to have a clinical benefit
and can be further tested for its ability to prevent cancer cell growth
in animal models or test subjects.

Selecting a Therapeutic Agent for Treating and/or Preventing NSCLC that
is Appropriate for a Particular Individual

[0225] Differences in the genetic makeup of individuals can result in
differences in their relative abilities to metabolize various drugs. A
compound that is metabolized in a subject to act as an anti-NSCLC agent
can manifest itself by inducing a change in gene expression pattern in
the subject's cells from that characteristic of a cancerous state to a
gene expression pattern characteristic of a non-cancerous state.
Accordingly, the differentially expressed KIF11, GHSR1b, NTSR1, and FOXM1
genes disclosed herein allow for selection of a putative therapeutic or
prophylactic inhibitor of NSCLC specifically adequate for a subject by
testing candidate compounds in a test cell (or test cell population)
derived from the selected subject.

[0226] To identify an anti-NSCLC agent, that is appropriate for a specific
subject, a test cell or test cell population derived from the subject is
exposed to a therapeutic agent and the expression of one or more of the
KIF11, GHSR1b, NTSR1, and FOXM1 genes is determined.

[0227] The test cell is or the test cell population contains an NSCLC cell
expressing an NSCLC-associated gene. Preferably, the test cell is or the
test cell population contains a lung cell. For example, the test cell or
test cell population is incubated in the presence of a candidate agent
and the pattern of gene expression of the test cell or cell population is
measured and compared to one or more reference profiles, e.g., an NSCLC
reference expression profile or an non-NSCLC reference expression
profile.

[0228] A decrease in the expression of one or more of KIF11, GHSR1b,
NTSR1, and FOXM1 in a test cell or test cell population relative to a
reference cell population containing NSCLC is indicative that the agent
is therapeutic.

[0229] The test agent can be any compound or composition. For example, the
test agent is an immunomodulatory agent.

Methods for Treating or Preventing NSCLC

[0230] The present invention provides a method for treating, alleviating
or preventing NSCLC in a subject. Therapeutic compounds are administered
prophylactically or therapeutically to subjects suffering from or at risk
of (or susceptible to) developing NSCLC. Such subjects are identified
using standard clinical methods or by detecting an aberrant level of
expression or activity of KIF11, GHSR1b, NTSR1 or FOXM1 gene or
polypeptide. Prophylactic administration occurs prior to the
manifestation of overt clinical symptoms of disease, such that a disease
or disorder is prevented or alternatively delayed in its progression.

[0231] The method includes decreasing the expression or function, or both,
of one or more gene products of genes whose expression is aberrantly
increased ("over-expressed gene"; KIF11, GHSR1b, NTSR1 or FOXM1 gene) in
an NSCLC cell relative to normal cells of the same tissue type from which
the NSCLC cells are derived. The expression may be inhibited by any
method known in the art. For example, a subject may be treated with an
effective amount of a compound that decreases the amount of one or more
of the KIF11, GHSR1b, NTSR1 or FOXM1 gene in the subject. Administration
of the compound can be systemic or local. Such therapeutic compounds
include compounds that decrease the expression level of such gene that
endogenously exists in the NSCLC cells (i.e., compounds that
down-regulate the expression of the over-expressed gene(s), KIF11, GHSR1b
and/or NTSR1 genes). The administration of such therapeutic compounds
counter the effects of aberrantly-over expressed gene(s) in the subjects
NSCLC cells and are expected to improve the clinical condition of the
subject. Such compounds can be obtained by the screening method of the
present invention described above.

[0232] The compounds that modulate the activity of a protein encoded by
KIF11, GHSR1b, NTSR1 or FOXM1 gene that can be used for treating or
preventing NSCLC of the present invention include besides proteins,
naturally-occurring cognate ligand of these proteins, peptides,
peptidomimetics and other small molecules.

[0233] Alternatively, the expression of these over-expressed gene(s)
(KIF11, GHSR1b, NTSR1 and/or FOXM1) can be inhibited by administering to
the subject a nucleic acid that inhibits or antagonizes the expression of
the over-expressed gene(s). Antisense oligonucleotides, siRNAs or
ribozymes which disrupt the expression of the over-expressed gene(s) can
be used for inhibiting the expression of the over-expressed gene(s).

[0234] As noted above, antisense-oligonucleotides corresponding to any of
the nucleotide sequence of KIF11, GHSR1b, NTSR1 or FOXM1 gene can be used
to reduce the expression level of the gene. Antisense-oligonucleotides
corresponding to KIF11, GHSR1b, NTSR1, and FOXM1 genes that are
up-regulated in NSCLC are useful for the treatment or prevention of
NSCLC. Specifically, the antisense-oligonucleotides against the genes may
act by binding to any of the corresponding polypeptides encoded by these
genes, or mRNAs corresponding thereto, thereby inhibiting the
transcription or translation of the genes, promoting the degradation of
the mRNAs, and/or inhibiting the expression of proteins encoded by the
KIF11, GHSR1b, NTSR1, and FOXM1 nucleotides, and finally inhibiting the
function of the proteins. The term "antisense-oligonucleotides" as used
herein encompasses both nucleotides that are entirely complementary to
the target sequence and those having a mismatch of one or more
nucleotides, so long as the antisense-oligonucleotides can specifically
hybridize to the target sequence. For example, the
antisense-oligonucleotides of the present invention include
polynucleotides that have a homology (also referred to as sequence
identity) of at least 70% or higher, preferably at 80% or higher, more
preferably 90% or higher, even more preferably 95% or higher over a span
of at least 15 continuous nucleotides up to the full length sequence of
any of the nucleotide sequences of KIF11, GHSR1b, NTSR1 or FOXM1 gene.
Algorithms known in the art can be used to determine the homology.
Furthermore, derivatives or modified products of the
antisense-oligonucleotides can also be used as antisense-oligonucleotides
in the present invention. Examples of such modified products include
lower alkyl phosphonate modifications such as methyl-phosphonate-type or
ethyl-phosphonate-type, phosphorothioate modifications and
phosphoroamidate modifications.

[0235] siRNA molecules of the invention can also be defined by their
ability to hybridize specifically to mRNA or cDNA from the genes
disclosed here. For the purposes of this invention the terms "hybridize"
or "hybridize specifically" are used to refer the ability of two nucleic
acid molecules to hybridize under "stringent hybridization conditions."
The phrase "stringent hybridization conditions" refers to conditions
under which a nucleic acid molecule will hybridize to its target
sequence, typically in a complex mixture of nucleic acids, but not
detectably to other sequences. Stringent conditions are
sequence-dependent and will be different in different circumstances.
Longer sequences hybridize specifically at higher temperatures. An
extensive guide to the hybridization of nucleic acids is found in
Tijssen, Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and the
strategy of nucleic acid assays" (1993). Generally, stringent conditions
are selected to be about 5-10° C. lower than the thermal melting
point (Tm) for the specific sequence at a defined ionic strength pH.
The Tm is the temperature (under defined ionic strength, pH, and
nucleic concentration) at which 50% of the probes complementary to the
target hybridize to the target sequence at equilibrium (as the target
sequences are present in excess, at Tm, 50% of the probes are
occupied at equilibrium). Stringent conditions may also be achieved with
the addition of destabilizing agents such as formamide. For selective or
specific hybridization, a positive signal is at least two times
background, preferably 10 times background hybridization. Exemplary
stringent hybridization conditions can be as following: 50% formamide,
5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1%
SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1%
SDS at 50° C. The antisense-oligonucleotides and derivatives
thereof act on cells producing the proteins encoded by KIF11, GHSR1b,
NTSR1 or FOXM1 gene by binding to the DNA or mRNA encoding the protein,
inhibiting transcription or translation thereof, promoting the
degradation of the mRNAs and inhibiting the expression of the protein,
thereby resulting in the inhibition of the protein function.

[0236] An antisense-oligonucleotides and derivatives thereof can be made
into an external preparation, such as a liniment or a poultice, by mixing
with a suitable base material which is inactive against the derivative.

[0237] The antisense-oligonucleotides of the invention inhibit the
expression of at least one protein encoded by any one of KIF11, GHSR1b,
NTSR1, and FOXM1 genes, and thus are useful for suppressing the
biological activity of the protein.

[0238] The polynucleotides that inhibit one or more gene products of
over-expressed genes also include small interfering RNAs (siRNA)
comprising a combination of a sense strand nucleic acid and an antisense
strand nucleic acid of the nucleotide sequence encoding an over-expressed
protein encoded by KIF11, GHSR1b, NTSR1 or FOXM1 gene. The term "siRNA"
refers to a double stranded RNA molecule which prevents translation of a
target mRNA. Standard techniques of introducing siRNA into the cell can
be used in the treatment or prevention of the present invention,
including those in which DNA is a template from which RNA is transcribed.
The siRNA is constructed such that a single transcript has both the sense
and complementary antisense sequences from the target gene, e.g., a
hairpin.

[0239] The method is used to suppress gene expression of a cell with
up-regulated expression of KIF11, GHSR1b, NTSR1 or FOXM1 gene. Binding of
the siRNA to KIF11, GHSR1b, NTSR1 or FOXM1 gene transcript in the target
cell results in a reduction of KIF11, GHSR1b, NTSR1 or FOXM1 protein
production by the cell. The length of the oligonucleotide is at least
about 10 nucleotides and may be as long as the naturally occurring
transcript. Preferably, the oligonucleotide is about 19 to about 25
nucleotides in length. Most preferably, the oligonucleotide is less than
about 75, about 50 or about 25 nucleotides in length. Preferable siRNA of
the present invention include the polynucleotides having the nucleotide
sequence of SEQ ID NO: 32, 33, 34, 35, 36, 37, or 108 as the target
sequence, which all proved to be effective for suppressing cell viability
of NSCLC cell lines. Specifically, a preferable siRNA used in the present
invention has the general formula:

5'-[A]-[B]-[A']-3'

wherein [A] is a ribonucleotide sequence corresponding to a target
sequence of KIF11, GHSR1b, NTSR1 or FOXM1; [B] is a ribonucleotide
sequence consisting of about 3 to about 23 nucleotides; and [A'] is a
ribonucleotide sequence complementary to [A]. Herein, the phrase a
"target sequence of KIF11, GHSR1b, NTSR1 or FOXM1 gene" refers to a
sequence that, when introduced into NSCLC cell lines, is effective for
suppressing cell viability. Preferred target sequence of KIF11, GHSR1b,
NTSR1 or FOXM1 gene includes nucleotide sequences comprising: SEQ ID NOs:
32, 33, 34, 35, 36, 37, and 108. The complementary sequence [A'] and [A]
hybridize to each other to form a double strand, and the whole siRNA
molecule with the general formula 5'-[A]-[B]-[A]-3' forms a hairpin loop
structure. As used herein, the term "complementary" refers to a
Watson-Crick or Hoogsteen base pairing between nucleotide units of a
polynucleotide, and hybridization or binding of nucleotide units
indicates physical or chemical interaction between the units under
appropriate conditions to form a stable duplex (double-stranded
configuration) containing few or no mismatches. In a preferred
embodiment, such duplexes contain no more than 1 mismatch for every 10
base pairs. Particularly preferred duplexes are fully complementary and
contain no mismatch. The siRNA against the mRNA of KIF11, GHSR1b, NTSR1
or FOXM1 gene to be used in the present invention contains a target
sequence shorter than the whole mRNA of KIF11, GHSR1b, NTSR1 or FOXM1
gene, and has a sequence of 500, 200, or 75 nucleotides as the whole
length. Also included in the invention is a vector containing one or more
of the nucleic acids described herein, and a cell containing the vectors.
The isolated nucleic acids of the present invention are useful for siRNA
against KIF11, GHSR1b, NTSR1 or FOXM1 or DNA encoding the siRNA. When the
nucleic acids are used for siRNA or coding DNA thereof, the sense strand
is preferably longer than about 19 nucleotides, and more preferably
longer than about 21 nucleotides.

[0240] Furthermore, the nucleotide sequence of siRNAs may be designed
using a siRNA design computer program available from the Ambion website
(found on the World Wide Web at
ambion.com/techlib/misc/siRNA_finder.html). The nucleotide sequences for
the siRNA are selected by the computer program based on the following
protocol:

Selection of siRNA Target Sites: [0241] 1. Beginning with the AUG start
codon of the transcript, scan downstream for AA dinucleotide sequences.
Record the occurrence of each AA and the 3' adjacent 19 nucleotides as
potential siRNA target sites. Tuschl, et al. Genes Dev 13(24): 3191-7
(1999), not recommend against designing siRNA against the 5' and 3'
untranslated regions (UTRs) and regions near the start codon (within 75
bases) as these may be richer in regulatory protein binding sites, and
thus the complex of endonuclease and siRNAs that were designed against
these regions may interfere with the binding of UTR-binding proteins
and/or translation initiation complexes. [0242] 2. Compare the potential
target sites to the human genome database and eliminate from
consideration any target sequences with significant homology to other
coding sequences. The homology search can be performed using BLAST, which
can be found on the NCBI server at: www.ncbi.nlm.nih.gov/BLAST/3. [0243]
3. Select qualifying target sequences for synthesis. On the website of
Ambion, several preferable target sequences can be selected along the
length of the gene for evaluation.

[0244] The siRNAs inhibit the expression of over-expressed KIF11, GHSR1b,
NTSR1 or FOXM1 protein and is thereby useful for suppressing the
biological activity of the protein. Therefore, a composition comprising
the siRNA is useful in treating or preventing non-small cell lung cancer.

[0245] The nucleic acids that inhibit one or more gene products of
over-expressed genes KIF11, GHSR1b, NTSR1, and FOXM1 also include
ribozymes against the gene(s).

[0246] The ribozymes inhibit the expression of over-expressed KIF11,
GHSR1b, NTSR1 or FOXM1 protein and is thereby useful for suppressing the
biological activity of the protein. Therefore, a composition comprising
the ribozyme is useful in treating or preventing NSCLC.

[0247] Generally, ribozymes are classified into large ribozymes and small
ribozymes. A large ribozyme is known as an enzyme that cleaves the
phosphate ester bond of nucleic acids. After the reaction with the large
ribozyme, the reacted site consists of a 5'-phosphate and 3'-hydroxyl
group. The large ribozyme is further classified into (1) group I intron
RNA catalyzing transesterification at the 5'-splice site by guanosine;
(2) group II intron RNA catalyzing self-splicing through a two step
reaction via lariat structure; and (3) RNA component of the ribonuclease
P that cleaves the tRNA precursor at the 5' site through hydrolysis. On
the other hand, small ribozymes have a smaller size (about 40 bp)
compared to the large ribozymes and cleave RNAs to generate a 5'-hydroxyl
group and a 2'-3' cyclic phosphate. Hammerhead type ribozymes (Koizumi et
al., FEBS Lett. 228: 225 (1988)) and hairpin type ribozymes (Buzayan,
Nature 323: 349 (1986); Kikuchi and Sasaki, Nucleic Acids Res. 19: 6751
(1992)) are included in the small ribozymes. Methods for designing and
constructing ribozymes are known in the art (see Koizumi et al., FEBS
Lett. 228: 225 (1988); Koizumi et al., Nucleic Acids Res. 17: 7059
(1989); Kikuchi and Sasaki, Nucleic Acids Res. 19: 6751 (1992)) and
ribozymes inhibiting the expression of an over-expressed NSC protein can
be constructed based on the sequence information of the nucleotide
sequence encoding KIF11, GHSR1b, NTSR1 or FOXM1 protein according to
conventional methods for producing ribozymes.

[0248] The ribozymes inhibit the expression of over-expressed KIF11,
GHSR1b, NTSR1 or FOXM1 protein and is thereby useful for suppressing the
biological activity of the protein. Therefore, a composition comprising
the ribozyme is useful in treating or preventing NSCLC.

[0249] Alternatively, the function of one or more gene products of the
over-expressed genes is inhibited by administering a compound that binds
to or otherwise inhibits the function of the gene products. For example,
the compound is an antibody which binds to the over-expressed gene
product or gene products.

[0250] The present invention refers to the use of antibodies, particularly
antibodies against a protein encoded by any of the up-regulated genes
KIF11, GHSR1b, NTSR1 or FOXM1, or a fragment of the antibody. As used
herein, the term "antibody" refers to an immunoglobulin molecule having a
specific structure that interacts (binds) specifically with a molecule
comprising the antigen used for synthesizing the antibody (i.e., the
up-regulated gene product) or with an antigen closely related to it. An
antibody that binds to the over-expressed KIF11, GHSR1b, NTSR1 or FOXM1
nucleotide may be in any form, such as monoclonal or polyclonal
antibodies, and includes antiserum obtained by immunizing an animal such
as a rabbit with the polypeptide, all classes of polyclonal and
monoclonal antibodies, human antibodies and humanized antibodies produced
by genetic recombination. Furthermore, the antibody used in the method of
treating or preventing NSCLC of the present invention may be a fragment
of an antibody or a modified antibody, so long as it binds to one or more
of the proteins encoded by the marker genes (KIF11, GHSR1b, NTSR1 or
FOXM1 gene). The antibodies and antibody fragments used in the present
method of treating or preventing NSCLC may be modified, and include
chemically modified and chimeric antibodies. Such antibodies and antibody
fragments can be obtained according to the above-mentioned methods,
supra.

[0251] When the obtained antibody is to be administered to the human body
(antibody treatment), a human antibody or a humanized antibody is
preferable for reducing immunogenicity.

[0252] For example, transgenic animals having a repertory of human
antibody genes may be immunized with an antigen such as KIF11, GHSR1b,
NTSR1 or FOXM1 polypeptide, cells expressing the polypeptide, or their
lysates. Antibody producing cells are then collected from the animals and
fused with myeloma cells to obtain hybridoma, from which human antibodies
against the polypeptide can be prepared (see WO92-03918, WO93-2227,
WO94-02602, WO94-25585, WO96-33735, and WO96-34096).

[0253] Alternatively, an immune cell, such as an immunized lymphocyte,
producing antibodies may be immortalized by an oncogene and used for
preparing monoclonal antibodies.

[0254] The present invention provides a method for treating or preventing
NSCLC, using an antibody against an over-expressed KIF11, GHSR1b, NTSR1
or FOXM1 polypeptide. According to the method, a pharmaceutically
effective amount of an antibody against KIF11, GHSR1b, NTSR1 or FOXM1
polypeptide is administered. An antibody against an over-expressed KIF11,
GHSR1b, NTSR1 or FOXM1 polypeptide is administered at a dosage sufficient
to reduce the activity of KIF11, GHSR1b, NTSR1 or FOXM1 protein.
Alternatively, an antibody binding to a cell surface marker specific for
tumor cells can be used as a tool for drug delivery. Thus, for example,
an antibody against an over-expressed KIF11, GHSR1b, NTSR1 or FOXM1
polypeptide conjugated with a cytotoxic agent may be administered at a
dosage sufficient to injure tumor cells.

[0255] In addition, dominant negative mutants of the proteins disclosed
here can be used to treat or prevent NSCLC. For example, fragments of
KOC1 that specifically bind KIF11 can be used. As used here "dominant
negative fragment of KOC1" is a mutated form of KOC1 that is capable of
complexing with either of KIF11 and RNA to be transported such that the
RNA transporter activity of the complex is diminished. Thus, a dominant
negative fragment is one that is not functionally equivalent to the full
length KOC1 polypeptide. Preferred dominant negative fragments are those
that comprise at least one RRM domain of KOC1. Alternatively, in another
embodiment, the dominant negative fragments have two RRM domains and zero
to three of KH domains. For example KOC1DEL2 (2×RRM+2×KH) and
KOC1DEL3 (2×RRM without KH) are preferable fragment for dominant
negative effect. The amino acid sequences of KOC1DEL2 and KOC1DEL3
consist of positions 1 to 406 and 1-197 of SEQ ID NO:105, respectively.
The fragments are typically less than about 300 amino acids, typically
less than about 200 amino acids.

[0256] The present invention also relates to a method of treating or
preventing NSCLC in a subject comprising administering to said subject a
vaccine comprising a polypeptide encoded by a nucleic acid selected from
the group consisting of KIF11, GHSR1b, NTSR1, and FOXM1 genes or an
immunologically active fragment of said polypeptide, or a polynucleotide
encoding the polypeptide or the fragment thereof. Administration of the
polypeptide induces an anti-tumor immunity in a subject. Thus, the
present invention further provides a method for inducing anti tumor
immunity. The polypeptide or the immunologically active fragments thereof
are useful as vaccines against NSCLC. In some cases the proteins or
fragments thereof may be administered in a form bound to the T cell
receptor (TCR) or presented on an antigen presenting cell (APC), such as
macrophage, dendritic cell (DC) or B-cells. Due to the strong antigen
presenting ability of DC, the use of DC is most preferable among the
APCs.

[0257] In the present invention, the phrase "vaccine against NSCLC" refers
to a substance that has the function to induce anti-tumor immunity or
immunity to suppress NSCLC upon inoculation into animals. In general,
anti-tumor immunity includes immune responses such as follows: [0258]
induction of cytotoxic lymphocytes against tumors, [0259] induction of
antibodies that recognize tumors, and [0260] induction of anti-tumor
cytokine production.

[0261] Therefore, when a certain protein induces any one of these immune
responses upon inoculation into an animal, the protein is decided to have
anti-tumor immunity inducing effect. The induction of the anti-tumor
immunity by a protein can be detected by observing in vivo or in vitro
the response of the immune system in the host against the protein.

[0262] For example, a method for detecting the induction of cytotoxic T
lymphocytes is well known. A foreign substance that enters the living
body is presented to T cells and B cells by the action of antigen
presenting cells (APCs). T cells that respond to the antigen presented by
APC in antigen specific manner differentiate into cytotoxic T cells (or
cytotoxic T lymphocytes; CTLs) due to stimulation by the antigen, and
then proliferate (this is referred to as activation of T cells).
Therefore, CTL induction by a certain peptide can be evaluated by
presenting the peptide to T cell by APC, and detecting the induction of
CTL. Furthermore, APC has the effect of activating CD4+ T cells, CD8+ T
cells, macrophages, eosinophils and NK cells. Since CD4+ T cells are also
important in anti-tumor immunity, the anti-tumor immunity inducing action
of the peptide can be evaluated using the activation effect of these
cells as indicators.

[0263] A method for evaluating the inducing action of CTL using dendritic
cells (DCs) as APC is well known in the art. DC is a representative APC
having the strongest CTL inducing action among APCs. In this method, the
test polypeptide is initially contacted with DC and then this DC is
contacted with T cells. Detection of T cells having cytotoxic effects
against the cells of interest after the contact with DC shows that the
test polypeptide has an activity of inducing the cytotoxic T cells.
Activity of CTL against tumors can be detected, for example, using the
lysis of 51Cr-labeled tumor cells as the indicator. Alternatively,
the method of evaluating the degree of tumor cell damage using
3H-thymidine uptake activity or LDH (lactose dehydrogenase)-release
as the indicator is also well known.

[0264] Apart from DC, peripheral blood mononuclear cells (PBMCs) may also
be used as the APC. The induction of CTL is reported to be enhanced by
culturing PBMC in the presence of GM-CSF and IL-4. Similarly, CTL is
shown to be induced by culturing PBMC in the presence of keyhole limpet
hemocyanin (KLH) and IL-7.

[0265] The test polypeptides confirmed to possess CTL inducing activity by
these methods are polypeptides having DC activation effect and subsequent
CTL inducing activity. Therefore, polypeptides that induce CTL against
tumor cells are useful as vaccines against NSCLC. Furthermore, APC that
acquired the ability to induce CTL against NSCLC by contacting with the
polypeptides are useful as vaccines against NSCLC. Furthermore, CTL that
acquired cytotoxicity due to presentation of the polypeptide antigens by
APC can be also used as vaccines against NSCLC. Such therapeutic methods
for NSCLC using anti-tumor immunity due to APC and CTL are referred to as
cellular immunotherapy.

[0266] Generally, when using a polypeptide for cellular immunotherapy,
efficiency of the CTL-induction is known to increase by combining a
plurality of polypeptides having different structures and contacting them
with DC. Therefore, when stimulating DC with protein fragments, it is
advantageous to use a mixture of multiple types of fragments.

[0267] Alternatively, the induction of anti-tumor immunity by a
polypeptide can be confirmed by observing the induction of antibody
production against tumors. For example, when antibodies against a
polypeptide are induced in a laboratory animal immunized with the
polypeptide, and when growth, proliferation or metastasis of tumor cells
is suppressed by those antibodies, the polypeptide can be determined to
have an ability to induce anti-tumor immunity.

[0268] Anti-tumor immunity is induced by administering the vaccine of this
invention, and the induction of anti-tumor immunity enables treatment and
prevention of NSCLC. Therapy against or prevention of the onset of NSCLC
includes any of the steps, such as inhibition of the growth of NSCLC
cells, involution of NSCLC cells and suppression of occurrence of NSCLC
cells. Decrease in mortality of individuals having NSCLC, decrease of
marker genes (in addition to KIF11, GHSR1b and/or NTSR1 genes) in the
blood, alleviation of detectable symptoms accompanying NSCLC and such are
also included in the therapy or prevention of NSCLC. Such therapeutic and
preventive effects are preferably statistically significant. For example,
in observation, at a significance level of 5% or less, wherein the
therapeutic or preventive effect of a vaccine against NSCLC is compared
to a control without vaccine administration. For example, Student's
t-test, the Mann-Whitney U-test or ANOVA may be used for statistical
analysis.

[0269] The above-mentioned protein having immunological activity, or a
polynucleotide or vector encoding the protein may be combined with an
adjuvant. An adjuvant refers to a compound that enhances the immune
response against the protein when administered together (or successively)
with the protein having immunological activity. Examples of adjuvants
include cholera toxin, salmonella toxin, alum and such, but are not
limited thereto. Furthermore, the vaccine of this invention may be
combined appropriately with a pharmaceutically acceptable carrier.
Examples of such carriers are sterilized water, physiological saline,
phosphate buffer, culture fluid and such. Furthermore, the vaccine may
contain as necessary, stabilizers, suspensions, preservatives,
surfactants and such. The vaccine is administered systemically or
locally. Vaccine administration may be performed by single administration
or boosted by multiple administrations.

[0270] When using APC or CTL as the vaccine of this invention, NSCLC can
be treated or prevented, for example, by the ex vivo method. More
specifically, PBMCs of the subject receiving treatment or prevention are
collected, the cells are contacted with the polypeptide ex vivo, and
following the induction of APC or CTL, the cells may be administered to
the subject. APC can be also induced by introducing a vector encoding the
polypeptide into PBMCs ex vivo. APC or CTL induced in vitro can be cloned
prior to administration. By cloning and growing cells having high
activity of damaging target cells, cellular immunotherapy can be
performed more effectively. Furthermore, APC and CTL isolated in this
manner may be used for cellular immunotherapy not only against
individuals from whom the cells are derived, but also against similar
types of diseases in other individuals.

[0271] Moreover, the present invention provides a method for treating or
preventing NSCLC in a subject, wherein a compound obtained according to
any of the above-described screening methods is administered to the
subject. Any compound that are obtained according to any of the screening
methods of the present invention can be administered to the subject so
long as it decreases the expression or function, or both, of one or more
gene products of KIF11, GHSR1b, NTSR1, and FOXM1 genes.

siRNA and Vectors Encoding Them

[0272] Transfection of vectors expressing siRNA for KIF11, GHSR1b, NTSR1
or FOXM1 leads to growth inhibition of NSCLC cells. Thus, it is another
aspect of the present invention to provide a double-stranded molecule
comprising a sense-strand and antisense-strand which molecule functions
as an siRNA for KIF11, GHSR1b, NTSR1 or FOXM1, and a vector encoding the
double-stranded molecule.

[0273] The double-stranded molecule of the present invention comprises a
sense strand and an antisense strand, wherein the sense strand comprises
a ribonucleotide sequence corresponding to a KIF11, GHSR1b, NTSR1 or
FOXM1 target sequence, and wherein the antisense strand comprises a
ribonucleotide sequence which is complementary to said sense strand,
wherein said sense strand and said antisense strand hybridize to each
other to form said double-stranded molecule, and wherein said
double-stranded molecule, when introduced into a cell expressing a KIF11,
GHSR1b, NTSR1 or FOXM1 gene, inhibits expression of said gene.

[0274] The double-stranded molecule of the present invention may be a
polynucleotide derived from its original environment (i.e., when it is a
naturally occurring molecule, the natural environment), physically or
chemically altered from its natural state, or chemically synthesized.
According to the present invention, such double-stranded molecules
include those composed of DNA, RNA, and derivatives thereof. A DNA is
suitably composed of bases such as A, T, C and G, and T is replaced by U
in an RNA.

[0275] As described above, the term "complementary" refers to a
Watson-Crick or Hoogsteen base pairing between nucleotide units of a
polynucleotide, and hybridization or binding of nucleotide units
indicates physical or chemical interaction between the units under
appropriate conditions to form a stable duplex (double-stranded
configuration) containing few or no mismatches. In a preferred
embodiment, such duplexes contain no more than 1 mismatch for every 10
base pairs. Particularly preferred duplexes are fully complementary and
contain no mismatch.

[0276] The double-stranded molecule of the present invention contains a
ribonucleotide sequence corresponding to a KIF11, GHSR1b, NTSR1 or FOXM1
target sequence shorter than the whole mRNA of KIF11, GHSR1b, NTSR1 or
FOXM1 gene. Herein, the phrase a "target sequence of KIF11, GHSR1b, NTSR1
or FOXM1 gene" refers to a sequence that, when introduced into NSCLC cell
lines, is effective for suppressing cell viability. Specifically, the
target sequence comprises at least about 10, or suitably about 19 to
about 25 contiguous nucleotides from the nucleotide sequences selected
from the group of SEQ ID NOs: 1, 3, 5, and 106. That is, the sense strand
of the present double-stranded molecule consists of at least about 10
nucleotides, suitably is longer than 19 nucleotides, and more preferably
longer than 21 nucleotides. Preferred target sequences include the
sequences of SEQ ID NOs: 32, 33, 34, 35, 36, 37, and 108. The present
double-stranded molecule including the sense strand and the antisense
strand is an oligonucleotide shorter than about 100, preferably 75, more
preferably 50 and most preferably 25 nucleotides in length. A suitable
double-stranded molecule of the present invention is an oligonucleotide
of a length of about 19 to about 25 nucleotides. Furthermore, in order to
enhance the inhibition activity of the siRNA, nucleotide "u" can be added
to 3' end of the antisense strand of the target sequence. The number of
"u"s to be added is at least 2, generally 2 to 10, preferably 2 to 5. The
added "u"s form single strand at the 3' end of the antisense strand of
the siRNA. In these embodiments, the siRNA molecules of the invention are
typically modified as described above for antisense molecules. Other
modifications are also possible, for example, cholesterol-conjugated
siRNAs have shown improved pharmacological properties (Song et al. Nature
Med. 9:347-351 (2003):).

[0277] Furthermore, the double-stranded molecule of the present invention
may be a single ribonucleotide transcript comprising the sense strand and
the antisense strand linked via a single-stranded ribonucleotide
sequence. Namely, the present double-stranded molecule may have the
general formula:

5'-[A]-[B]-[A']-3'

wherein [A] is a ribonucleotide sequence corresponding to a target
sequence of KIF11, GHSR1b, NTSR1 or FOXM1; [B] is a ribonucleotide
sequence (loop sequence) consisting of 3 to 23 nucleotides; and [A] is a
ribonucleotide sequence complementary to [A]. The complementary sequence
[A] and [A] hybridize to each other to form a double strand, and the
whole siRNA molecule with the general formula 5'-[A]-[B]-[A']-3' forms a
hairpin loop structure.

[0278] The region [A] hybridizes to [A], and then a loop consisting of
region [B] is formed. The loop sequence can be selected from those
described on the World Wide Web at
ambion.com/techlib/tb/tb--506.html, or those described in Jacque,
J.-M. et al., Nature 418: 435-438 (2002). Additional examples of the loop
sequence that can be included in the present double-stranded molecules
include:

[0280] Preferable siRNAs having hairpin loop structure of the present
invention are shown below. In the following structure, the loop sequence
can be selected from the group consisting of: CCC, UUCG, CCACC, CCACACC,
and UUCAAGAGA. Among these sequences, the most preferable loop sequence
is UUCAAGAGA (corresponding to "ttcaagaga" in a DNA):

[0281] The present invention further provides a vector encoding the
double-stranded molecule of the present invention. The vector encodes a
transcript having a secondary structure and which comprises the sense
strand and the antisense strand, and suitably comprises a single-stranded
ribonucleotide sequence linking said sense strand and said antisense
strand. The vector preferably comprises a regulatory sequence adjacent to
the region encoding the present double-stranded molecule that directs the
expression of the molecule in an adequate cell. For example, the
double-stranded molecules of the present invention are intracellularly
transcribed by cloning their coding sequence into a vector containing,
e.g., a RNA pol III transcription unit from the small nuclear RNA (snRNA)
U6 or the human H1 RNA promoter.

[0282] Alternatively, the present vectors are produced, for example, by
cloning the target sequence into an expression vector so the objective
sequence is operatively-linked to a regulatory sequence of the vector in
a manner to allow expression thereof (transcription of the DNA molecule)
(Lee, N. S. et al., Nature Biotechnology 20: 500-505 (2002)). For
example, the transcription of an RNA molecule having an antisense
sequence to the target sequence is driven by a first promoter (e.g., a
promoter sequence linked to the 3'-end of the cloned DNA) and that having
the sense strand to the target sequence by a second promoter (e.g., a
promoter sequence linked to the 5'-end of the cloned DNA). The expressed
sense and antisense strands hybridize to each other in vivo to generate a
siRNA construct to silence a gene that comprises the target sequence.
Furthermore, two constructs (vectors) may be utilized to respectively
produce the sense and anti-sense strands of a siRNA construct.

[0283] For introducing the vectors into a cell, transfection-enhancing
agent can be used. FuGENE (Rochediagnostices), Lipofectamine 2000
(Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pure
Chemical) are useful as the transfection-enhancing agent.

Pharmaceutical Compositions for Treating or Preventing NSCLC

[0284] The present invention provides compositions for treating or
preventing NSCLC comprising a compound selected by the present method of
screening for a compound that alters the expression or activity of an
NSCLC-associated gene.

[0285] When administering a compound isolated by the screening method of
the present invention as a pharmaceutical for humans and other mammals,
such as mice, rats, guinea-pig, rabbits, cats, dogs, sheep, pigs, cattle,
monkeys, baboons or chimpanzees for treating a cell proliferative disease
(e.g., non-small cell lung cancer), the isolated compound can be directly
administered or can be formulated into a dosage form using conventional
pharmaceutical preparation methods. Such pharmaceutical formulations of
the present compositions include those suitable for oral, rectal, nasal,
topical (including buccal and sub-lingual), vaginal or parenteral
(including intramuscular, sub-cutaneous and intravenous) administration,
or for administration by inhalation or insufflation. The formulations are
optionally packaged in discrete dosage units.

[0286] Pharmaceutical formulations suitable for oral administration
include capsules, cachets or tablets, each containing a predetermined
amount of the active ingredient. Formulations also include powders,
granules, solutions, suspensions or emulsions. The active ingredient is
optionally administered as a bolus electuary or paste. Tablets and
capsules for oral administration may contain conventional excipients such
as binding agents, fillers, lubricants, disintegrant or wetting agents. A
tablet may be made by compression or molding, optionally with one or more
formulational ingredients. Compressed tablets may be prepared by
compressing in a suitable machine the active ingredients in a
free-flowing form such as powder or granules, optionally mixed with a
binder, lubricant, inert diluent, lubricating, surface active or
dispersing agent. Molded tablets may be made via molding in a suitable
machine a mixture of the powdered compound moistened with an inert liquid
diluent. The tablets may be coated according to methods well known in the
art. Oral fluid preparations may be in the form of, for example, aqueous
or oily suspensions, solutions, emulsions, syrups or elixirs, or may be
presented as a dry product for reconstitution with water or other
suitable vehicle prior to use. Such liquid preparations may contain
conventional additives such as suspending agents, emulsifying agents,
non-aqueous vehicles (which may include edible oils) or preservatives.
The tablets may optionally be formulated so as to provide slow or
controlled release of the active ingredient in vivo. A package of tablets
may contain one tablet to be taken on each of the month. The formulation
or dose of medicament in these preparations makes a suitable dosage
within the indicated range acquirable.

[0287] Formulations for parenteral administration include aqueous and
non-aqueous sterile injection solutions which may contain anti-oxidants,
buffers, bacteriostats and solutes which render the formulation isotonic
with the blood of the intended recipient; and aqueous and non-aqueous
sterile suspensions which may include suspending agents and thickening
agents. The formulations may be presented in unit dose or multi-dose
containers, for example sealed ampoules and vials, and may be stored in a
freeze-dried (lyophilized) condition requiring only the addition of the
sterile liquid carrier, for example, saline, water-for-injection,
immediately prior to use. Alternatively, the formulations may be
presented for continuous infusion. Extemporaneous injection solutions and
suspensions may be prepared from sterile powders, granules and tablets of
the kind previously described.

[0288] Formulations for rectal administration include suppositories with
standard carriers such as cocoa butter or polyethylene glycol.
Formulations for topical administration in the mouth, for example,
buccally or sublingually, include lozenges, which contain the active
ingredient in a flavored base such as sucrose and acacia or tragacanth,
and pastilles comprising the active ingredient in a base such as gelatin,
glycerin, sucrose or acacia. For intra-nasal administration of an active
ingredient, a liquid spray or dispersible powder or in the form of drops
may be used. Drops may be formulated with an aqueous or non-aqueous base
also comprising one or more dispersing agents, solubilizing agents or
suspending agents.

[0289] For administration by inhalation the compositions are conveniently
delivered from an insufflator, nebulizer, pressurized packs or other
convenient means of delivering an aerosol spray. Pressurized packs may
comprise a suitable propellant such as dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or
other suitable gas. In the case of a pressurized aerosol, the dosage unit
may be determined by providing a valve to deliver a metered amount.

[0290] Alternatively, for administration by inhalation or insufflation,
the compositions may take the form of a dry powder composition, for
example, a powder mix of an active ingredient and a suitable powder base
such as lactose or starch. The powder composition may be presented in
unit dosage form in, for example, capsules, cartridges, gelatin or
blister packs from which the powder may be administered with the aid of
an inhalator or insufflators.

[0291] Other formulations include implantable devices and adhesive
patches; which release a therapeutic agent.

[0292] When desired, the above-described formulations, adapted to give
sustained release of the active ingredient, may be employed. The
pharmaceutical compositions may also contain other active ingredients
such as antimicrobial agents, immunosuppressants or preservatives.

[0293] It should be understood that in addition to the ingredients
particularly mentioned above, the formulations of this invention may
include other agents conventional in the art having regard to the type of
formulation in question, for example, those suitable for oral
administration may include flavoring agents.

[0294] Preferred unit dosage formulations are those containing an
effective dose, as recited below, of the active ingredient or an
appropriate fraction thereof.

[0295] For each of the aforementioned conditions, the compositions, e.g.,
polypeptides and organic compounds are administered orally or via
injection at a dose of from about 0.1 to about 250 mg/kg per day. The
dose range for adult humans is generally from about 5 mg to about 17.5
g/day, preferably about 5 mg to about 10 g/day, and most preferably about
100 mg to about 3 g/day. Tablets or other unit dosage forms of
presentation provided in discrete units may conveniently contain an
amount which is effective at such dosage or as a multiple of the same,
for instance, units containing about 5 mg to about 500 mg, usually from
about 100 mg to about 500 mg.

[0296] The dose employed will depend upon a number of factors, including
the age and sex of the subject, the precise disorder being treated, and
its severity. Also the route of administration may vary depending upon
the condition and its severity.

[0297] The present invention further provides a composition for treating
or preventing NSCLC comprising active ingredient that inhibits the
expression of any one of the gene selected from the group of KIF11,
GHSR1b, NTSR1, and FOXM1 genes. Such active ingredient can be an
antisense-oligonucleotide, siRNA or ribozyme against the gene, or
derivatives, such as expression vector, of the antisense-oligonucleotide,
siRNA or ribozyme. The active ingredient may be made into an external
preparation, such as liniment or a poultice, by mixing with a suitable
base material which is inactive against the derivatives.

[0299] Preferably, the antisense-oligonucleotide derivative, siRNA
derivative or ribozyme derivative is given to the patient by direct
application to the ailing site or by injection into a blood vessel so
that it will reach the site of ailment. A mounting medium can also be
used in the composition to increase durability and membrane-permiability.
Examples of mounting mediums include liposome, poly-L-lysine, lipid,
cholesterol, lipofectin and derivatives thereof.

[0300] The dosage of such compositions can be adjusted suitably according
to the patient's condition and used in desired amounts. For example, a
dose range of 0.1 to 100 mg/kg, preferably 0.1 to 50 mg/kg can be
administered.

[0301] Another embodiment of the present invention is a composition for
treating or preventing NSCLC comprising an antibody against a polypeptide
encoded by any one of the genes selected from the group of KIF11, GHSR1b,
NTSR1, and FOXM1 genes or fragments of the antibody that bind to the
polypeptide.

[0302] Although there are some differences according to the symptoms, the
dose of an antibody or fragments thereof for treating or preventing NSCLC
is about 0.1 mg to about 100 mg per day, preferably about 1.0 mg to about
50 mg per day and more preferably about 1.0 mg to about 20 mg per day,
when administered orally to a normal adult (weight 60 kg).

[0303] When administering parenterally, in the form of an injection to a
normal adult (weight 60 kg), although there are some differences
according to the condition of the patient, symptoms of the disease and
method of administration, it is convenient to intravenously inject a dose
of about 0.01 mg to about 30 mg per day, preferably about 0.1 to about 20
mg per day and more preferably about 0.1 to about 10 mg per day. Also, in
the case of other animals too, it is possible to administer an amount
converted to 60 kg of body-weight.

[0304] The following examples are presented to illustrate the present
invention and to assist one of ordinary skill in making and using the
same. The examples are not intended in any way to otherwise limit the
scope of the invention.

[0305] Unless otherwise defined, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. Although methods and
materials similar or equivalent to those described herein can be used in
the practice or testing of the present invention, suitable methods and
materials are described below. Any patents, patent applications and
publications sited herein are incorporated by reference.

[0309] Total RNA was extracted from cultured cells and clinical tissues
using Trizol reagent (Life Technologies, Inc.) according to the
manufacturer's protocol. Extracted RNAs and normal human tissue poly(A)
RNAs were treated with DNase I (Nippon Gene) and reverse-transcribed
using oligo(dT) primer and SuperScript II reverse transcriptase
(Invitrogen). Semiquantitative RT-PCR experiments were carried out with
the following synthesized gene-specific primers or with beta-actin
(ACTB)-specific primers as an internal control:

[0311] Expression levels of the KOC1 and KIF11 genes were measured by
QRT-PCR using the ABI Prism 7700 sequence detection system (Applied
Biosystems). Total RNA was extracted from cultured cells and clinical
tissues using Trizol reagent (Life Technologies, Inc.) according to the
manufacturer's protocol. Extracted RNAs and normal human tissue poly(A)
RNAs were treated with DNase I (Nippon Gene) and were reverse-transcribed
using oligo (dT) primer and SuperScript II reverse transcriptase
(Invitrogen). The TaqMan Pre-Developed Assay Human ACTB (Applied
Biosystems; #4333762F) was used for ACTB gene as a quantitative control.
A primer pair and a TaqMan probe for each gene were designed by using
Primer Express software as follows:

[0312] PCR for each gene and the ACTB gene was performed in a single tube
in duplicate. Results were expressed as the average of these two
independent tests.

(4) Northern-Blot Analysis

[0313] Human multiple-tissue blots (BD Biosciences Clontech) were
hybridized with 32P-labeled PCR products of KOC1, KIF11 and GHSR1.
cDNA probes of KOC1, KIF11 and GHSR1 were prepared by RT-PCR using
primers similarly as above. Prehybridization, hybridization, and washing
were performed according to the supplier's recommendations. The blots
were autoradiographed with intensifying BAS screens (BIO-RAD) at room
temperature (RT) for 30 to 168 hours.

Generation of Anti-KOC1 and -KIF11 Antibodies

[0314] Plasmids expressing KOC1 (full-length) and KIF11 (partial amino
acid sequence corresponding to codons 361-1056), each containing
His-tagged epitope at the N-terminal, were prepared using pET28 vector
(Novagen). Recombinant proteins were expressed in Escherichia coli BL21
codon-plus strain (Stratagene), purified using TALON resin (BD
Biosciences Clontech) according to the supplier's protocol, and
inoculated into rabbits. The immune sera were purified on affinity
columns according to standard methodology. Affinity-purified anti-KOC1
and anti-KIF11 antibodies were used for western-blot analysis,
immunoprecipitation, and immunostaining We confirmed by western-blot
analysis that anti-KOC1 antibody are specific to KOC1 and do not
cross-react with other homologous proteins, IMP-1 and IMP-2 using lysates
of NCI-H520 cells, which expressed neither of endogenous IMP-1, -2, and
-3, but had been transfected with HA-tagged IMP-1, -2, and -3 expression
vector.

Construction of KOC1 Deletion Mutants and Immunoprecipitation Assays for
Identification of the KOC1-KIF11 Binding Region

[0315] KOC1 and several of its domains (FIG. 3a) were cloned into
appropriate sites of N-terminal FLAG-tagged and C-terminal HA-tagged
pCAGGS vector. COS-7 cells transfected only with an KOC1 deletion mutant,
were immunoprecipitated with anti-HA agarose (SIGMA). Endogenous KIF11
bands were detected with affinity-purified anti-KIF11 antibody by western
blotting.

RNA-immunoprecipitation and cDNA Microarray Screening for Identification
of KOC1-Associated mRNAs

[0316] We adopted the RNA immunoprecipitation protocol of Niranjanakumari
et al. (Niranjanakumari, S. et al. Methods 26, 182-190 (2002)) to analyze
RNA-protein interactions involving KOC1 in vivo. Immunoprecipitated RNAs
were isolated from five lung-cancer cell lines (A549, LC319, PC14,
RERF-LC-AI, and SK-MES-1). A 2.5-μg aliquot of T7-based amplified RNAs
(aRNAs) from each immunoprecipiated RNA (IP-RNA) and from the total RNA
(control) were reversely transcribed in the presence of Cy5-dCTP and
Cy3-dCTP respectively as described previously (Kikuchi, T. et al.
Oncogene 22, 2192-2205 (2003)), for hybridization to a cDNA microarray
representing 32,256 genes (IP-microarray analysis). To confirm the
binding to KOC1 of the mRNAs identified by IP-microarray analysis, we
carried out RT-PCR experiments using gene-specific primers and RNAs from
NSCLC cell extracts immunoprecipitated with anti-KOC1 antibody
(IP-RT-PCR). To confirm the region of KOC1 required for binding to the
KOC1-associated mRNAs, we also carried out northwestern blot analysis as
below and IP-RT-PCR of KOC1-associated mRNAs from these
immunoprecipitated extracts transfected with various KOC1 deletion
mutants.

[0319] In vitro transcription of linearized plasmids carrying the
full-length cDNA sequence of an KOC1-associated gene, RAB35, was
performed using DAVIS Lab's protocol (found on the World Wide Web at
ed.ac.uk/˜ilan). To generate fluorescent riboprobes for in vivo
co-localization with KOC1, the plasmids were transcribed using the mCAP
RNA capping kit (Stratagene) in the presence of Alexa Fluor 546-labeled
UTP (Molecular Probes). We constructed plasmids expressing EGFP-fused
KOC1 (EGFP-KOC1) protein were prepared using pEGFP-N1 vectors (BD
Biosciences Clontech). For live-cell imaging of co-localized EGFP-KOC1
and Alexa Fluor 546-labeled RAB35 mRNA, COS-7 cells that had been
transfected initially with pEGFP-KOC1 were additionally transfected 36
hours later with Alexa Fluor 546-labeled RAB35 mRNA (3 μg per 3.5-cm
culture dish) in the presence of RNase Inhibitor (TAKARA). The
plasmid-DNA and RNA samples were transfected using Lipofectamine 2000
(Invitrogen) according to the manufacturer's protocols. The cells were
washed twice with PBS, and fresh medium was added 90 min after
transfection with the labeled mRNA. The cells were allowed to recover in
the incubator (37° C., 5% CO2) for 30 min before live-cell
imaging for 3-6 hours with a confocal microscope (FV1000 FLUOVIEW,
OLYMPUS). To investigate the specific transport of mRNAs by KOC1-RNP
complex from one cell to another cell, we prepared two different
COS-7-derived cells; the COS-7 cells transfected with pEGFP-KOC1 and
Alexa Fluor 546-labeled RAB35 mRNA and the other, parental COS-7 cells
simply labeled with CellTracker (Molecular Probes) according to the
supplier's protocols. These two cell populations were mixed and
co-cultured for 12 hours before live-cell imaging with confocal
microscope for 6 hours.

[0320] To investigate the translation of the mRNA transported by KOC1-RNP
complex in the recipient cells, we prepared two types of COS-7-derived
cell; one type was COS-7 cells co-transfected with pCAGGS-FLAG
tagged-KOC1 and -KIF11. After 24 hours culture, plasmid containing
EGFP-fused RAB35 full length mRNA were re-transfected into these cells.
The other type was COS-7 cells simply labeled with CellTracker (blue).
These two cell-types were mixed and co-cultured for 24 hours before
live-cell imaging with video microscope for 12 hours. Synthesis of
EGFP-tagged RAB35 mRNAs and corresponding proteins in the
CellTracker-stained recipient cells (blue) as well as on the ultrafine
structure between the two cells was examined by in situ hybridization and
time-lapse video microscopy.

Fluorescent In Situ Hybridization

[0321] We carried out in situ hybridization with DIG-labeled probes
complementary to RAB35 or EGFP mRNA at 60° C. for 16 hours. The
DIG label was detected using NBT-BCIP, an alkaline phosphatase color
substrate. Cells were washed, mounted and visualized on light microscope.
Fixed cells were hybridized with a mixture of DIG-labeled complementary
to RAB35 mRNA for 16 hours in 50% formamide/2×SSC at 42° C.
Cells were washed, mounted and visualized on confocal microscope.

(5) RNA Interference Assay

[0322] To prepare plasmid vector expressing short interfering RNA (siRNA),
we amplified the genomic fragment of H1RNA gene containing its promoter
region by PCR using a set of primers, 5'-TGGTAGCCAAGTGCAGGTTATA-3' (SEQ
ID No: 44), and 5'-CCAAAGGGTTTCTGCAGTTTCA-3' (SEQ ID No: 45) and human
placental DNA as a template. The product was purified and cloned into
pCR2.0 plasmid vector using a TA cloning kit according to the supplier's
protocol (Invitrogen). The BamHI and XhoI fragment containing H1RNA was
into pcDNA3.1(+) between nucleotides 1257 and 56, and the fragment was
amplified by PCR using

[0323] 5'-TGCGGATCCAGAGCAGATTGTACTGAGAGT-3' (SEQ ID No: 46) and

[0324] 5'-CTCTATCTCGAGTGAGGCGGAAAGAACCA-3' (SEQ ID No: 47),

[0325] The ligated DNA became the template for PCR amplification with
primers,

[0326] 5'-TTTAAGCTTGAAGACCATTTTTGGAAAAAAAAAAAC-3' (SEQ ID No: 48) and

[0327] 5'-TTTAAGCTTGAAGACATGGGAAAGAGTGGTCTCA-3' (SEQ ID No: 49).

[0328] The product was digested with HindIII, and subsequently
self-ligated to produce psiH1BX3.0 vector plasmid having a nucleotide
sequence shown in SEQ ID NO: 50.

[0329] The DNA fragment encoding siRNA was inserted into the GAP at
nucleotide 489-492 as indicated (-) in the following plasmid sequence
(SEQ ID NO: 50).

[0331] The oligonucleotides used for these siRNAs are shown below. Each
constructs were prepared by cloning the following double-stranded
oligonucleotide into the BbsI site in the psiH1BX3.0 vector. The
corresponding nucleotide position relative to the KIF11, GHSR1b, NTSR1,
RAB35 and FOXM1 nucleic acid sequence of SEQ ID NOs:1, 3, 5, 112, and 106
are listed for each oligonucleotide sequence. Each oligonucleotide is a
combination of a sense nucleotide sequence and an antisense nucleotide
sequence of the target sequence of KIF11, GHSR1b, NTSR1, RAB35 and FOXM1.
The nucleotide sequences of the hairpin loop structure of each siRNAs are
also shown bellow. (endonuclease recognition sites are eliminated from
each hairpin loop structure sequence).

[0332] To validate RNAi system of the present invention, individual
control siRNAs (EGFP, Luciferase, and Scramble) were initially confirmed
using semiquantitative RT-PCR to decrease the expression of the
corresponding target genes that had been transiently transfected into
COS-7 cells. Down-regulation of KIF11, GHSR1b, NTSR1, RAB35 and FOXM1
expression by their respective siRNAs (si-KIF11-1, si-KIF11-2,
si-KIF11-3, si-GHSR-1, si-NTSR1-1, si-NTSR1-2, si-RAB35 and si-FOXM1),
but not by controls, was confirmed with semiquantitative RT-PCR in the
cell lines used for this assay.

Dominant-Negative Assays

[0333] We performed dominant-negative assays using the KOC1 deletion
mutants to investigate the functional role of the KOC1-KIF11 complex in
growth or survival of lung-cancer cells. The KOC1DEL3 and KOC1DEL2
construct (FIG. 3a; 10 μg), mock plasmid (10 μg), or plasmid
mixtures of both constructs in the final dose of 10-μg DNA (KOC1DEL3
or KOC1DEL2 vs mock (μg), 7.5:2.5; 5:5; or 2.5:7.5, individually) were
transfected into LC319 cells. The transfected cells were cultured for 7
days in the presence of G418 and their viability was measured by
triplicate MTT assays.

(6) Co-Immunoprecipitation and MALDI-TOF Mass Spectrometry

[0334] Human lung cancer cell line LC319 cells were transfected with
bilateral-tagged pCAGGS-n3FH (NH2-terminal FLAG, COOH-terminal HA)-KOC1
expression vector or empty vector (mock transfection). Cells were
extracted in IP-buffer (0.5% NP-40, 50 mM Tris-HCl, 150 mM NaCl, and
protease inhibitor) for 30 min on ice. Extracts were centrifuged at
14,000 rpm for 15 min, and supernatants were subjected to
immunoprecipitation using anti-Flag M2 agarose (Sigma-Aldrich) and
anti-HA beads (Sigma-Aldrich) for 1-2 hours. The beads were washed six
times with IP-buffer, and protein was eluted by boiling the beads in
Laemmli sample buffer after removing the final wash fraction. The eluted
protein was resolved by SDS-PAGE and stained with silver staining. A 125
kDa-band was extracted by gel extraction, and used for mass spectrometric
sequencing using MALDI-TOF mass spectrometry. This analysis identified
KIF11 as the 125 kDa product.

[0335] To confirm the interaction between KOC1 and KIF11, A549 cells were
transiently co-transfected with Flag-tagged KIF11 and myc-tagged KOC1 and
the cells were immunoprecipitated with anti-Flag M2 agarose.
Subsequently, the cells were immunoblotted with anti-myc antibody (9E10;
Santa Cruz). Next, using the same combination of vectors and cells, the
cells were immunoprecipitated with anti-myc agarose (SIGMA) and
immunoblotted with anti-Flag M2 antibody (Sigma-Aldrich).

[0336] To further confirm this interaction, A549 cells were transiently
co-transfected with Flag-tagged KIF11 and myc-tagged KOC1, and
co-localization of FITC-labeled KIF11 and rhodamine-labeled KOC1 mainly
in the cytoplasm was detected by immunocytochemical staining using
FITC-labeled anti-FLAG antibody and rhodamine-labeled anti-myc antibody,
as described below.

(7) Immunocytochemistry

[0337] A549 cells grown on coverslips were cultured for 24 hours after
passage, and were co-transfected with Flag-tagged KIF11 and myc-tagged
KOC1. After 24-hours incubation, the cells were fixed with
acetone/methanol (1:1) for 5 min on ice, blocked in CAS BLOCK (ZYMED) for
7 min at RT, and then incubated with rabbit anti-Flag polyclonal antibody
(SIGMA) for 1 hour at RT. The fixed cells were washed 3 times with PBS,
reacted with anti-rabbit IgG-FITC for 1 hour at RT. Then the cells were
blocked again, and incubated with anti-myc antibody (9E10; Santa Cruz)
for 1 hour at RT. Finally anti-mouse IgG-rhodamin was applied to the
cells for 1 hour at RT. The cells were viewed on a Leica TCS SP2-AOBS
confocal microscope.

[0339] To identify direct binding of NMU-25 to its candidate receptors,
GHSR1a, GHSR1b and NTSR1, the following experiments were performed. The
entire coding region of each receptor gene was amplified by RT-PCR using
primers

[0340] The products were digested with EcoRI and BamHI and cloned into
appropriate sites of p3XFLAG-CMV10 vector (Sigma-Aldrich Co.). COS-7
cells were transfected with GHSR1b or NTSR1 expression plasmids using
FuGENE6, as described above. Transfected COS-7 cells were cultured with
0.5 μM rhodamine-labeled NMU-25 peptide (NMU-25-rhodamine: Phoenix
Pharmaceuticals. Inc.) for 12 hours, washed five times in PBS(-), and
fixed in 4% paraformaldehyde solution for 60 min at room temperature.
Then the cells were incubated with antibodies to FLAG-tagged GHSR1a,
GHSR1b, or NTSR1 proteins (Sigma-Aldrich Co.), stained with a goat
anti-mouse secondary antibody conjugated to FITC (Cappel) and viewed
under laser-confocal microscopy (TCS SP2 AOBS: Leica Microsystems). In
addition, three different negative controls were prepared for this assay:
1) non-transfected COS-7 cells without addition of NMU-25-rhodamine; 2)
non-transfected COS-7 cells treated with NMU-25-rhodamine; and 3) COS-7
cells transfected with GHSR1a, GHSR1, or NTSR1 without NMU-25-rhodamine.
COS-7 cells transfected with a known NMU receptor (NMU1R) served as a
positive control for the assay.

[0341] To confirm the binding of NMU-25 to the candidate receptors,
flow-cytometric analysis was performed using the same series of COS-7
cells. Specifically, COS-7 cells were plated at a density of 1×105
cells/100-mm dish and transfected with either GHSR1b, NTSR1, or NMU1R
expression vectors; 24 hours after transfection, cells were incubated
with 0.5 μM NMU-25-rhodamine for 12 hours, washed, trypsinized,
collected in PBS, and washed once more in PBS. The population of cells
binding to rhodamine-labeled NMU-25 was determined by flow cytometry.

[0342] To further confirm binding of NMU-25 to the endogenous candidate
receptors on the NSCLC cells, we performed receptor-ligand binding assay
using the LC319 and PC-14 cells. Briefly, these cells trypsinized were
seeded onto 96-well black-wall, clear-bottom microtiter plates 24 hours
prior to the assay. The medium was removed and the cells were incubated
with Cy5-NMU-25 with a 10-fold excess of unlabeled competitor. The plate
was incubated in the dark for 24 hours at 37° C. and was scanned
on the 8200 Cellular Detection System (Applied Biosystems). 8200 Analysis
Software creates a digitized gray scale image, quantitates the amount of
fluorescence bound on the surface of each cell, and enumerates the
fluorescent cells.

[0346] Expression of the candidate genes was additionally detected by
semiquantitative RT-PCR using mRNAs isolated at 72 and 96 hours from
LC319 cells treated with 1 μM NMU-25 or BSA at the time point of 0 and
48 hours.

Results

[0347] (1) Identification of KIF11 as a Protein Interacting with KOC1

[0348] LC319 cells transfected with pCAGGS-n3FH-KOC1 vector were extracted
and immunoprecipitated with anti-Flag M2 monoclonal antibody, and
subsequently immunoprecipitated with anti-HA monoclonal antibody. The
protein complex including KOC1 was stained with silver staining on
SDS-PAGE gel. A 125 kDa band that was absent in mock transfection was
extracted and determined to be KIF11 (NM--004523; SEQ. ID. NO. 1) by
Mass spectrometric sequencing.

(2) Confirmation of Interaction Between KOC1 and KIF11

[0349] The A549 cells co-transfected with Flag-tagged KIF11 and myc-tagged
KOC1, the cells transfected with either KIF11 or KOC1, and the
non-transfected cells were immunoprecipitated with anti-Flag M2 agarose
and subsequently immunoblotted with anti-myc antibody. In contrast, the
same series of A549 cells were immunoprecipitated with anti-myc agarose
and immunoblotted with anti-Flag M2 antibody. A single band was found
only when both constructs were co-transfected (FIG. 1a).
Immunocytochemistry showed that FLAG-tagged FITC-labeled KIF11
co-localized in cytoplasm of A549 with myc-tagged rhomamine-labeled KOC1
(FIG. 1b).

[0350] Next, we confirmed by western blot analysis that anti-KOC1 antibody
are specific to KOC1 and do not cross-react with other homologous
proteins, IMP-1 and IMP-2 using H520 cell lysate, which had been
confirmed to be not expressed endogenous IMP-1, -2, and -3 (KOC1), but
had been transfected with HA-tagged IMP-1, -2, and -3 (KOC1) expression
vector. Lysates of LC319 cells transfected with pCAGGS-FLAG-tagged-KOC1
vector or mock vector (control) were extracted and immunoprecipitated
with anti-FLAG M2 monoclonal antibody. The protein complex including KOC1
was stained with SilverQuest (Invitrogen) on an SDS-PAGE gel. A 125-kDa
band was detected specifically in immunoprecipitates from lysates of
cells transfected with KOC1 expressing plasmids, but not in control
lysates (mock plasmids). Subsequent MALDI-TOF mass spectrometric analysis
identified this 125-kDa protein as KIF11, a member of the kinesin family.
We confirmed direct interaction between endogenous KOC1 and KIF11 by
immunoprecipitation experiments with extracts from A549 and LC319, using
affinity-purified anti-KOC1 and anti-KIF11 polyclonal antibodies (FIG.
1c).

(3) KIF11 Expression in NSCLC

[0351] Validation of KIF11 expression was performed in primary NSCLCs
(clinical samples) and lung cancer cell lines. Increased KIF11 expression
was confirmed in 12 of 16 NSCLC cases (5 of 8 ADCs and in 7 of 8 SCCs. In
addition, up-regulation of KIF11 were observed in 14 of the 15 NSCLC cell
lines.

[0352] We subsequently re-examined primary NSCLC tissues and lung-cancer
cell lines, and found increased KIF11 expression in 18 NSCLC clinical
samples as well as in all of the 20 NSCLC or SCLC cell lines examined by
quantitative RT-PCR (FIG. 2a,b). The mRNA levels of the KOC1 and KIF11
genes relative to ACTB genes were significantly correlated (r=0.702,
P=0.0029 by the Spearman rank correlation). These two genes were
coactivated in almost lung cancer cell lines (r=0.458, P=0.0359 by the
Spearman rank correlation).

(4) KIF11 Expression in Normal Human Tissues

[0353] Northern blotting with KIF11 cDNA as a probe identified 4.5- and
5.5-kb transcripts as very weak bands, only seen in placenta, testis, and
bone marrow, among the 23 normal human tissues examined. The reported
cDNA sequence of KIF11 was considered to correspond to the larger
transcript. To investigate the transcript corresponding to the smaller
band, we reversely transcribed mRNAs isolated from tissues of the testis
and NSCLC cell lines. We amplified the entire sequence of KIF11 cDNA by
PCR using four primer sets, but found no alternatively-spliced transcript
in these samples. Therefore, the smaller band may reflect
cross-hybridization to the transcript of some related gene(s). The
expression pattern of KIF11 in normal human tissues was significantly
correlated with that of KOC1 (FIG. 2c).

Identification of the KIF11-Binding Region in KOC1

[0354] To determine the specific domain of KOC1 required for interaction
with KIF11, we transfected into COS-7 cells one of five
deletion-constructs of KOC1 with NH2 (N)-terminal FLAG- or COOH
(C)-terminal HA-tagged sequences (KOC1DEL1-5; FIG. 3a).
Immunoprecipitation with monoclonal anti-HA indicated that the KOC1DEL4
and KOC1DEL5 constructs, which both lacked two RNA-recognition motifs
(RRMs), were unable to interact with endogenous KIF11, while all KOC1
constructs possessing the two RRMs retained binding affinity for KIF11
(FIG. 3b).

[0355] Isolation of mRNAs Associated with the KOC1-KIF11 Complex Using
RNA-Immunoprecipitation and cDNA Microarray

[0356] KOC1 protein is known to exhibit multiple attachments to IGF2
leader-3 mRNA, possibly through its two functional RRMs and four
K-homologous (KH) domains (Nielsen, J. et al., Mol. Cell Biol. 19,
1262-1270 (1999).). However, we did not detect expression of IGF2 mRNA in
any of the lung-cancer cell lines or clinical NSCLC tissue samples we
examined. Therefore, to elucidate the function of KOC1 in lung
carcinogenesis, we searched for mRNA(s) that would interact with KOC1 and
might thereby play important roles in growth and/or progression of lung
cancer. First we immunoprecipitated mRNAs using anti-KOC1 antibody and
five NSCLC cell lines (A549, LC319, PC14, RERF-LC-AI, and SK-MES-1).
Then, Cy-5-labeled immunoprecipitated RNAs (IP-mRNA) and Cy-3-labeled
total RNAs isolated from each matching cell line, were co-hybridized on
human cDNA microarrays (IP-microarray). Among 32,256 genes screened, we
identified a total of 55 that were enriched in IP-mRNA compared with
total RNA in at least four of the five NSCLC cell lines tested (see Table
2), and confirmed enrichment of all those candidates by RT-PCR using the
IP-mRNAs as templates (IP-RT-PCR). To examine the specificity of
RNA-immunoprecipitation, we performed RT-PCR experiments with beta-actin
(ACTB) mRNA using IP-mRNA as template; no ACTB was precipitated by
anti-KOC1 antibody. As background controls of RNA-immunoprecipitation, we
precipitated mRNAs using normal rabbit IgG and five NSCLC cell lines, and
confirmed that none of eight KOC1 associated mRNAs tested (CCT2, SBP2,
SLC25A3, RAB35, PSMB7, GL, PKP4, and WINS1) was precipitated by normal
rabbit IgG. We also confirmed elevated expression of many of the
candidate genes in NSCLC samples by RT-PCR (data not shown). To examine
whether the KOC1-KIF11 complex formation requires the co-presence of
these KOC1-associated mRNAs, we performed immunoprecipitation experiments
using cell lysates which were treated or untreated in vitro with 30 units
of RNase T1 (SIGMA), and found no difference in the interaction of the
two proteins in the presence or absence of mRNAs, suggesting that the
KOC1-KIF11 complex formation is unlikely to require these specific mRNAs.

[0357] By pursuing that strategy we have been able to show that KOC1 and
KIF11 not only are co-over-expressed in the great majority of clinical
NSCLC samples and cell lines, but also that a complex formed by the
products of these genes is indispensable for growth and progression of
NSCLC cells, by contributing to an intra- and inter-cellular
mRNA-transporting system. Intracellular mRNA transport by RNA-binding
proteins has been reported in oocytes and developing embryos of
Drosophila and Xenopus, and in somatic cells such as fibroblasts and
neurons (King, M. L. et al., Bioessays 21, 546-557 (1999); Mowry, K. L. &
Cote, C. A. Faseb. J. 13, 435-445 (1999); Lasko, P., J. Cell Biol. 150,
F51-56 (2000); Steward, O. Neuron 18, 9-12 (1997)) beta-actin mRNA is
transported to the leading lamellae of chicken-embryo fibroblasts (CEFs)
and to the growth cones of developing neurons (Lawrence, J. B. & Singer,
R. H. Cell 45, 407-415 (1986); Bassell, G. J. et al., J. Neurosci. 18,
251-265 (1998)). The localization of ACTB mRNA depends on the "zipcode",
a cis-acting element in the 3'UTR of the mRNA (Kislauskis, E. H. et al.,
J. Cell Biol. 123, 165-172 (1993)). The respective trans-acting factor,
zipcode-binding protein 1 (ZBP1), was identified by affinity purification
with the zipcode of ACTB mRNA; (Ross, A. F. et al., Mol. Cell Biol. 17,
2158-2165 (1997)) homologues of ZBP1 have since been identified in a wide
range of organisms including Xenopus, Drosophila, mouse, and human
(Mueller-Pillasch, F. et al., Oncogene 14, 2729-2733 (1997); Deshler, J.
O. et al., Science 276, 1128-1131 (1997); Doyle, G. A. et al., Nucleic
Acids Res. 26, 5036-5044 (1998)). ZBP1-like proteins contain two RRMs in
the N-terminal region and four hnRNP KH (ribonucleoprotein K-homology)
domains at the C-terminal end. KOC1, one of the IGF2 mRNA-binding
proteins, is considered to be a member of the ZBP1 family; it exhibits
multiple attachments to IGF2 leader-3 mRNA (Nielsen, J. et al., Mol. Cell
Biol. 19, 1262-1270 (1999)) and is over-expressed in several types of
cancers (Mueller-Pillasch, F. et al., Oncogene 14, 2729-2733 (1997);
Zhang, J. Y. et al., Clin. Immunol. 100, 149-156 (2001); Mueller, F. et
al., Br. J. Cancer 88, 699-701 (2003); Wang, T. et al., Br. J. Cancer 88,
887-894 (2003)). However, since we failed to detect expression of IGF2
leader-3 mRNA in most of the NSCLC cell lines or clinical samples we
examined, we suspected that KOC1 could mediate growth of lung-cancer
cells through interaction with, and transport of, other mRNA(s). When we
undertook RNA-immunoprecipitation experiments coupled with cDNA
microarrays (IP-microarray), we identified dozens of candidate mRNAs that
were likely to be associated with KOC1 in NSCLC cells (see Table 2). That
list included genes encoding proteins with functions of cell-adhesion
(PKP4, L1CAM1), cancer-cell progression and invasion (IGFBP2), and
binding of small GTPs (RAB35), (Papagerakis, S. et al., Hum. Pathol. 34,
565-572 (2003); Fogel, M. et al., Cancer Lett. 189, 237-247 (2003); Wang,
H. et al., Cancer Res. 63, 4315-4321 (2003); Zhao, H. et al., Biochem.
Biophys. Res. Commun. 293, 1060-1065 (2002)) and many of them were
expressed at high levels in clinical NSCLC samples (data not shown).
Activation of a system that transports mRNAs whose products are
associated with growth or movement of cells is very interesting, and
further investigations along this line could lead to a better
understanding of pulmonary carcinogenesis.

[0358] To determine the region of KOC1 that is required for binding to
KOC1-associated mRNAs, we performed northwestern blot analysis using
immunoprecipitated recombinant proteins of KOC1 deletion-mutants
expressed in A549 cells (FIG. 4a) and DIG-labeled RAB35 mRNA, which is
one of the KOC1 associated mRNAs. The KOC1DEL3, which lacked four KH
domains, and KOC1DEL5, which lacked N-terminal two RRMs and C-terminal
two KH domains, did not bind to the RAB35 mRNA. On the other hand, the
KOC1DEL4, which is a construct with only the four KH domains and the
KOC1DEL2, a construct without C-terminal two KH domains showed very weak
binding affinities for mRNAs compared to the full-length KOC1 construct
(FIG. 4b), suggesting the importance of two RRMs as well as of C-terminal
two KH domains for binding to KOC1-associated mRNAs.

[0359] We further expressed five of the KOC1 deletion-mutants in A549
cells and performed immunoprecipitation experiments twice with the cell
lysates, first with monoclonal anti-HA and then with monoclonal anti-FLAG
M2 antibody. Using IP-mRNA, we examined the ability of each
deleted-protein to bind to eight endogenous mRNAs (CCT2, SBP2, SLC25A3,
RAB35, PSMB7, GL, PKP4, and WINS1) selected from the above list (see
Table 2). The results were completely concordant to that of northwestern
blot analysis, independently confirming that both C-terminal two KH
domains and two RRMs in the N-terminal are indispensable for effective
binding of KOC1 to mRNAs (FIG. 4c).

[0361] To further investigate the functional roles of KOC1 and KIF11, we
prepared plasmids designed to express ECFP-KOC1 (cyan) and EYFP-KIF11
(yellow). We then transfected the two plasmids together into COS-7 cells,
and examined their localization using immunofluorescence video-microscopy
and real-time confocal microscopy. Cells expressing both KOC1 and KIF11
protruded into the processes, and then connected with adjacent cells
(data not shown). A more detailed observation of living cells found that
the KOC1 had formed a complex with KIF11 (KOC1-KIF11 RNP complex; green
particle) that was transported from one cell to another through an
ultrafine structure connecting the two cells (FIG. 5a). Movement of the
KOC1-KIF11 complex appeared to be unidirectional from one cell to
another.

[0362] Furthermore, to examine whether KOC1-KIF11 complex could
specifically transport KOC1-associated-mRNAs from one cell having a high
level of KOC1-RNP complex to another having a lower level of the complex,
we mixed and co-cultured two different cell populations; one is COS-7
cells that had been transfected with pEGFP-KOC1 (green) as well as Alexa
Fluor 546-labeled full-length RAB35 mRNA (red), and the other is parental
COS-7 cells simply labeled with CellTracker (blue). We observed that not
only KOC1 particles (green), but also RNP particles of KOC1-RAB35 mRNA
(yellow) were transferred through the ultrafine structure from the former
cells to the latter ones (FIG. 5b). Using in situ hybridization on A549
cells in which both KOC1 and KIF11 were over-expressed, we further
confirmed that the endogenous RAB35 mRNA (green) localized on the
ultrafine intercellular structures as well as in the cytoplasm (data not
shown).

[0363] We also investigated the endogenous location of KOC1 and KIF11
particles on the ultrafine structure of microtubules bridging individual
A549 cells by an immunocytochemical study, using affinity-purified
anti-KOC1- or anti-KIF11 for primary antibody and Alexa594-labeled
anti-rabbit IgG for secondary antibody (Molecular Probe) and
anti-alpha-tubulin-FITC monoclonal antibody. A549 cells treated with 10
μM of the microtubule disrupting agent nocodazole (SIGMA) for four
hours showed collapse and aggregation of endogenous KOC1 and KIF11, along
with the depolymerization of microtubules in the cytoplasm. Moreover, no
particle was found on the residual structure between the cells. The
result suggested the possibility of a microtubule-dependent transporting
mechanism involving the KOC1-KIF11 complex. To further clarify the
detailed mechanism by which the KOC1-KIF11 complex transports mRNAs in
NSCLC cells, we have searched for other component(s) that might be
interacting with KIF11. Immunoprecipitation with anti-KIF11 polyclonal
antibody using a lysate of LC319 cells identified a 150-kDa protein,
which was later determined to be a dynactin 1 (DCTN1; p150, glued
homolog, Drosophila) by MALDI-TOF mass-spectrometric analysis. DCTN1 is
the largest subunit of DCTN, which binds to the cytoplasmic motor-protein
dynein and activates vesicle transport along microtubules (Holzbaur, E.
L. & Tokito, M. K. Genomics 31, 398-399 (1996); Tokito, M. K. et al.,
Mol. Biol. Cell 7, 1167-1180 (1996)), or binds to KIF11 to probably
participate in centrosome separation (Blangy, A. et al., J. Biol. Chem.
272, 19418-19424 (1997)). We observed endogenous co-localization of
KOC1/KIF11 and DCTN1 on the ultrafine structure between the individual
A549 cells by immunocytochemistry, using the combination of
affinity-purified anti-KOC1- or anti-KIF11-polyclonal antibodies for
primary antibody and Alexa488-labeled anti-rabbit IgG for secondary
antibody, and the combination of anti-DCTN1 monoclonal antibody (BD
transduction Laboratories, #610473) for primary antibody and
anti-Alexa594-labeled anti-rabbit IgG for secondary antibody. And we
confirmed direct interaction between endogenous KIF11 and DCTN1 by
immunoprecipitation experiments with extracts from A549 and LC319 cells,
using anti-KIF11 polyclonal antibody and anti-DCTN1 monoclonal antibody
(BD transduction Laboratories, #610473) (FIG. 6a).

[0364] To further demonstrate the KIF11-dependent intercellular transport
of mRNA, we examined the effect of monastrol, the cell-permeable
inhibitor that specifically inhibits the KIF11. Previous reports
indicated that monastrol partially inhibits KIF11 ATPase activity through
binding directly to the motor domain (DeBonis, S. et al., Biochemistry
42, 338-349 (2003); Kononen, J. et al., Nat. Med. 4, 844-847 (1998)).
Treatment of A549 cells with 150 μM monastrol (SIGMA) for 24 hours
induced the accumulation of endogenous KIF11 and exogenous EYFP-KIF11 at
the center of monoaster along microtubules and the cell cycle arrest in
mitosis with monopolar spindles, which is called "monoastral spindle".
Treatment of A549 cells with 150 μM of monastrol for 24 hours induced
cell cycle arrest for mitotic cells with monopolar spindles that is
called "monoastral spindle" and also caused accumulation of endogenous
KIF11 at the center of monoaster along microtubules. On the other hand,
most non-mitotic cells lost protrusion into the processes and then lost
connection to adjacent cells within 2-hour of the monastrol treatment.
Further quantitative analysis by counting the number of intercellular
ultrafine structures (n=252 structures) with time-lapse video-microscopy
demonstrated that more than a half of the cell-to-cell connections in
non-mitotic cells tested disappeared by the one-hour monastrol treatment.
However, six hours after the release of the cells from the monastrol
exposure, the intercellular bridge formation was re-constituted and cells
at normal mitosis process was observed, indicating that KIF11 was
indispensable for the formation of ultrafine intercellular structures
(data not shown).

[0365] Some cells lost protrusion into the processes and then did not
connected with adjacent cells. A more detailed observation of living
cells found that no KOC1-KIF11 RNP complex (green particle) was
transported from one cell to another through an ultrafine structure
connecting the two cells, which subsequently disappeared during
observation.

[0366] In this study we demonstrated endogenous interaction of KOC1, KIF11
and DCTN1 in human lung cancers, and revealed a possible role of those
complexes in transport of mRNAs from one cell to another. DCTN1, the
largest subunit of DCTN, binds to the cytoplasmic motor protein dynein
and activates vesicle transport along microtubules (Holzbaur, E. L. &
Tokito, M. K. Genomics 31, 398-399 (1996)). Dynein-DCTN interaction is
probably a key component of the mechanism of axonal transport of vesicles
and organelles (Holzbaur, E. L. & Tokito, M. K. Genomics 31, 398-399
(1996); Tokito, M. K. et al., Mol. Biol. Cell 7, 1167-1180 (1996)). The
binding of DCTN to dynein is reportedly critical for neuronal function,
since antibodies that specifically disrupt this binding block vesicle
motility along microtubules. In vitro interaction of DCTN1 and KIF11, and
their co-localization during mitosis have been observed (Blangy, A. et
al., J. Biol. Chem. 272, 19418-19424 (1997)), but no report has shown an
intercellular transporting system involving this complex. Since in our
experiments KIF11, a member of the kinesin family, was over-expressed in
NSCLCs along with KOC1, we suggest that direct interaction of KOC1,
KIF11, and DCTN1 could play a significant role in establishing specific
alignment of microtubules between lung-cancer cells.

Protein Synthesis by Transported KOC1-Associated mRNAs in the Receiving
Cells

[0367] To elucidate whether the mRNA transport by KOC1-KIF11 RNP complex
is physiologically relevant (the recipient cell can synthesize the
protein by translating the mRNAs transported), we constructed an
expression vector of full length RAB35 mRNA, one of the binding targets
of the KOC1/KIF11 complex, fused in frame to myc tagged and an EGFP
protein sequences. We then investigated whether this chimeric mRNA could
be transportable from one cell to another and subsequently translated
into the protein production in the recipient cell. FLAG-tagged KOC1 and
KIF11 expressing-COST cells were transfected with constructs with these
RAB35 mRNA-expressing construct (cell A). Parental mRNA-recipient COS-7
cells were simply stained with CellTracker (blue; cell B). These two cell
populations were mixed together and co-cultured for 24 hours. We first
confirmed the intercellular transportation of RAB35-EGFP mRNAs between
cells A and B by in situ hybridization using antisense EGFP as a probe;
after co-culture of the cells for 24 hours, weak-staining of RAB35-EGFP
mRNAs were detected in the CellTracker-stained cell B as well as on the
ultrafine structure between the two cell types (FIG. 7a). We then
examined a presence of the EGFP-fused RAB35 proteins in the
CellTracker-stained B-type cells were found using immunocytochemistry and
time-lapse video microscopy, respectively (FIGS. 7b and 7c). During these
observations using time-lapse video microscopy, no visible EGFP-protein
particle was transported from the type-A to type-B cells, but the EGFP
protein gradually appeared in the apparatus of cytoplasm, which seemed to
be endoplasmic reticulum (ER) of in the type-B cells (FIG. 7d). These
results have indicated that KOC1 and KIF11 should functionally associate
with a subset of mRNAs, which encode proteins possibly inducing cell
proliferation and/or adhesion, and that the presence of KOC1 and KIF11 is
indispensable to the cell-to-cell transportation. Although previous
reports suggested that high KOC1 levels might interfere with translation
of bound mRNAs such as IGF2 leader-3, our experiment of co-transfecting
KOC1 and full-length RAB35-EGFP mRNA constructs together into COS-7 cells
detected no decrease of RAB35-EGFP-fused protein levels (FIG. 7e).

[0368] Our experiments also revealed formation of protruding processes
connecting adjacent cells, and showed predominant co-distribution of
transfected RAB35 mRNAs and KOC1 protein on ultra-fine intercellular
structures in two lung-cancer cell lines (A549 and LC319) that expressed
high levels of endogenous KOC1 and KIF11. On the other hand, we did not
find specific localization of transfected RAB35 mRNAs in NCI-H520 cells,
which express KIF11 but not KOC1. That observation supported the
importance of co-activation of KOC1 and KIF11 for communication among
cancer cells. Among the known cell-to-cell communication systems in human
cancers, formation of functional gap junctions between malignant glioma
cells and vascular endothelial cells appears to influence angiogenesis in
the tumors (Zhang, W. et al., Cancer Res. 59, 1994-2003 (1999); Zhang, W.
et al., J. Neurosurg. 98, 846-853 (2003)). However, to our knowledge ours
is the first report to describe inter-cellular transport of mRNA by means
of ribonucleoprotein particles combined with motor proteins in mammalian
somatic cells and to assess its biological significance for formation of
an inter-cellular network critical for growth and survival of cancer
cells.

(5) Inhibition of Growth of NSCLC Cells by siRNA Against KIF11

[0369] Transfection of either siRNA plasmids for KIF11 into A549 (FIG. 8a)
or LC319 (data not shown) cells suppressed mRNA expression of the KIF11
in comparison to cells containing any of the three control siRNAs and
mock transfection. In accordance with the reduced mRNA expression, A549
and LC319 cells showed significant decreases in cell viability and colony
numbers measured by MTT (FIG. 8b) and colony-formation assays (data not
shown). We also investigated the effect by siRNA against KIF11 on
intercellular transport using time-lapse videoscopy. A similar phenomenon
to monastrol treatment was observed; some cells reduced protrusion into
the processes and the disappearance of the ultrafine structure connecting
the two cells.

[0370] To investigate the functional significance of KOC1-KIF11
interaction for growth or survival of lung-cancer cells, a deletion
fragment of KOC1 containing the two RRMs, which was able to interact with
KIF11 (KOC1DEL3; FIG. 3a, b) was examined for a dominant-negative
function of suppressing direct interaction between endogenous KOC1 and
KIF11. We transfected KOC1DEL3 and mock plasmid (control) into LC319
cells and detected interaction of KOC1DEL3 with endogenous KIF11. We
further verified that overexpression of the RRM domains reduce complex
formation between KOC1 and KIF11 by immunoprecipitation (FIG. 9a,b).
Expectedly, transfection of that fragment resulted in significant
dose-dependent decreases in cell viability as measured by MTT assay
(P<0.001, KOC1DEL3 vs mock; FIG. 9c). We also confirmed that
transfection of construct containing only KH-domains control have no
effect on proliferation.

[0371] Furthermore, to investigate the functional significance of
KOC1-KIF11 interaction for growth or survival of lung-cancer cells, a
deletion fragment of KOC1, which lacked the C-terminal two KH-domains
indispensable for mRNA binding but was able to interact with KIF11
(KOC1DEL2; FIG. 3a, b), was examined for a dominant-negative function of
suppressing direct interaction between endogenous KOC1 and KIF11. We
transfected KOC1DEL2 and mock plasmid (control) into A549 cells and
detected interaction of KOC1DEL2 with endogenous KIF11 (FIG. 9d). We
further verified by immunoprecipitation that over-expression of the
KOC1DEL2 reduced complex formation between endogenous KOC1 and KIF11
(FIG. 9e). Expectedly, transfection of the dominant-negative fragment
resulted in significant dose-dependent decreases in cell viability as
measured by MTT assay (P=0.0006, KOC1DEL2 vs mock; FIG. 9f).

[0372] We also examined some biological role(s) of these
KIF11-transporting mRNAs in controlling the cell growth or survival of
lung-cancer cells, we constructed plasmid to express siRNA against RAB35
(si-RAB35), which was identified as the KOC1-RNP complex-associated
mRNAs. Transfection of the plasmids (si-RAB35) into A549 cells
significantly suppressed expression of endogenous RAB35 in comparison
with the controls, and resulted in significant decreases in cell
viability and colony numbers measured by MTT and colony-formation assays
(FIG. 10a,b).

Association of KOC1 and KIF11 Over-Expression with Poor Prognosis of
NSCLC Patients

[0373] We performed immunohistochemical analysis with anti-KOC1 and
anti-KIF11 polyclonal antibodies using tissue microarrays consisting of
265 NSCLC tissues (FIG. 11a). Of the 265 cases, KOC1 staining was
positive for 172 (64.9%); 129 cases were positive for KIF11 (48.7%). The
expression pattern of KOC1 was significantly concordant with KIF11
expression in these tumors (X2=60.8, P<0.0001). We then asked
whether KOC1 and/or KIF11 over-expression could be associated with
clinical outcome. We found that expression of KOC1 in NSCLCs was
significantly associated with pT factor status (X2=23.1,
P<0.0001) and with tumor-specific 5-year survival (P=0.0115 by the
Log-rank test) (FIG. 11b, upper panel). Expression of KIF11 in NSCLCs was
significantly associated with pT factor (X2=15.0, P<0.0001), pN
factor (X2=4.4, P=0.0356), and 5 year-survival (P=0.0008 by the
Log-rank test) (FIG. 11b, lower panel). By univariate analysis pT, pN,
gender, and KOC1/KIF11 expression were each significantly related to a
poor tumor-specific survival among NSCLC patients. Furthermore, KOC1 and
KIF11 were determined to be independent prognostic factors by
multivariate analysis using a Cox proportional-hazard model (P=0.0499 and
P=0.0259, respectively).

[0375] Since NMU2R and NMU1R were originally isolated as homologues of
known neuropeptide GPCRs, unidentified NMU receptor(s) were speculated to
be members of the same family that would show some degree of homology to
NMU1R/NMU2R. Hence, candidate NMU receptors were searched using the BLAST
program. The results and their high expression levels in NSCLCs in the
expression profile data of the present inventors indicated GHSR1b
(NM--004122; SEQ ID NOs: 3 and 4) and NTSR1 (NM--002531; SEQ ID
NOs: 5 and 6) to be good candidates. GHSR has two transcripts, types 1a
and 1b. The full-length human type 1a cDNA encodes a predicted
polypeptide of 366 amino acids with seven transmembrane domains, a
typical feature of G protein-coupled receptors. A single intron divides
its open reading frame into two exons encoding transmembrane domains 1-5
and 6-7, thus placing the GHSR1a into the intron-containing class of
GPCRs. Type 1b is a non-spliced mRNA variant transcribed from a single
exon that encodes a polypeptide of 289 amino acids with five
transmembrane domains. The semiquantitative RT-PCR analysis using
specific primers for each variant indicated that GHSR1a was not expressed
in NSCLCs. On the other hand, GHSR1b and NTSR1 were expressed at a
relatively high level in some NSCLC cell lines, but not at all in normal
lung (FIG. 13a). The GHSR1b product has 46% homology to NMU1R, and NTSR1
encodes 418 amino acids with 47% homology to NMU1R.

(7) Identification of Candidate Receptors for NMU in NSCLC

[0376] To confirm direct interaction between NMU and GHSR1b/NTSR1, COS-7
cells were transiently transfected with plasmids designed to express
FLAG-tagged GHSR1b or NTSR1, and cultured in the presence of
rhodamine-labeled NMU-25. Then the localization of FLAG-tagged
GHSR1b/NTSR1 and NMU-25-rhodamine in the cells were examined using
anti-FLAG antibodies conjugated to FITC, and found that NMU-25 and either
of both receptors were located together on the cell membrane (FIG. 13c).
Co-localization of NMU-25 with these receptors was not observed in
control assays involving either of the following ligand/cell
combinations: 1) NMU-25-rhodamine incubated with COS-7 cells that were
not transfected with either of the receptor plasmids; 2) non-transfected
COS-7 cells incubated without NMU-25-rhodamine; and 3) COS-7 cells
transfected with either of the receptor plasmids, but incubated without
NMU-25-rhodamine. The result was confirmed by flow cytometry, which
revealed binding of rhodamine-labeled NMU-25 to the surface of COS-7
cells that expressed either of the two receptors (FIG. 13d) and binding
of rhodamine-labeled NMU-25 to the surface of COS-7 cells in a dose
dependent manner.

(8) GHSR1b Expression in Normal Human Tissues

[0377] As the expression of GHSR1b in normal human tissues was not
precisely reported at the time, the distribution of GHSR1b was determined
using human multiple tissue Northern-blot. Northern blotting with GHSR1b
cDNA as a probe identified a 0.9-kb transcript as a very weak signal band
in comparison with a 1.1-kb transcript GHSR1a, seen in the heart, liver,
skeletal muscle, pancreas, and stomach, among the 23 normal human tissues
examined (FIG. 13b).

[0378] To further confirm binding of NMU-25 to the endogenous GHSR1b and
NTSR1 on the NSCLC cells, we performed receptor-ligand binding assay
using the LC319 and PC-14 cells treated with NMU-25. We detected binding
of Cy5-labeled NMU-25 to the surface of these two cell lines that
expressed both of the two receptors, but scarcely expressed NMU1R/NMU2R
(FIG. 13e).

[0379] Biologically active ligands for GPCRs have been reported to bind
specifically to their cognate receptors and cause an increase in
second-messengers such as intracellular-Ca2+ and cAMP levels. We
therefore determined the ability of NMU to induce these second-messengers
in LC319 cells through its interaction with GHSR1b/NTSR1. cAMP
production, but not Ca2+ flux in LC319 cells, which express both
GHSR1b and NTSR1 was observed in a NMU-25 dose dependent manner, when the
cells were cultured in the presence of NMU-25 at final concentrations of
3-100 μM in the culture media. The results demonstrate that NSCLC
cells express functional GHSR1b/NTSR1 (FIG. 13f left panel). This effect
was confirmed to be NMU-25 specific by adding other reported ligands for
GHSR1b/NTSR1, GHRL or NTS (FIG. 13f right panel). In addition, GHRL and
NTS caused the mobilization response of intracellular calcium in LC319
cells (data not shown), suggesting a variety of function for the poorly
understood for GHSR1b and/or NTSR1.

(9) Inhibition of Growth of NSCLC Cells by siRNA Against GHSR/NTSR1

[0380] Furthermore, the biological significance of the NMU-receptor
interaction in pulmonary carcinogenesis was examined using plasmids
designed to express siRNA against GHSR or NTSR1 (si-GHSR-1, si-NTSR1-1,
and si-NTSR1-2). Transfection of either of these plasmids into A549 or
LC319 cells suppressed expression of the endogenous receptor in
comparison to cells containing any of the three control siRNAs (FIG.
14a). In accordance with the reduced expression of the receptors, A549
and LC319 cells showed significant decreases in cell viability (FIG. 14b)
and numbers of colonies (data not shown). These results strongly
supported the possibility that NMU, by interaction with GHSR1b and NTSR1,
might play a very significant role in development/progression of NSCLC.

Identification of Downstream Genes of NMU

[0381] To further elucidate the NMU-signaling pathway and identify
downstream genes regulated by NMU, siRNA against NMU (si-NMU) or LUC
(control siRNA) were transfected into LC319 cells which had overexpressed
NMU and down-regulations in gene expression were monitored using a cDNA
microarray that contained 32,256 genes. Among hundreds of genes detected
by this method, we performed Self-organizing map (SOM) clustering
analysis to further select candidate genes. SOM clustering is data mining
and visualization method originally developed by Kohonen (Kohonen, T.
(1990). The self-organizing map. IEEE 78, 1464-1480.) and applied to the
analysis of gene expression data from microarrays. The clustering method
is similar to k-means clustering (Kaech, S. M., et al., (2002). Cell 111,
837-851.) but differs in that genes are divided into groups based on
expression patterns, and relationships between groups are illustrated by
two-dimensional maps. The genes passing our variation filter were grouped
by a 5×4 SOM.

[0382] We initially selected 70 genes using SOM cluster analysis, whose
intensity were significantly decreased in accordance with the reduction
of NMU expression (FIG. 15a). Semiquantitative RT-PCR analysis confirmed
reduction of candidate transcripts in a time-dependent manner in LC319
cells transfected with si-NMU, but not with control siRNA for LUC (FIG.
15b). These transcripts were also confirmed to be up-regulated greater
than 2-fold in LC319 cells expressing exogenous NMU, compared with that
of normal lung tissues. Overexpression of these genes in accordance with
NMU expression were evaluated as well in lung-cancer tissues and cell
lines (data not shown). We finally identified 6 candidate NMU target
genes, which satisfied the above selection criteria; FOXM1, FLJ42024,
GCDH, CDK5RAP1, LOC134145, and NUP188 (FIG. 15b).

[0383] FOXM1 mRNA levels were significantly elevated in lung cancers
compared with normal lung tissues and its expression showed good
concordance with NMU and two receptors for NMU, GHSR1b and NTSR1, whereas
the function of FOXM1 in lung carcinogenesis remains unclear. Therefore,
we chose FOXM1 for further analysis. To determine specific induction of
the FOXM1 by the NMU ligand-receptor signaling, LC319 cells expressing
GHSR1b and NTSR1 were cultured in the presence of NMU-25 or BSA (control)
at final concentrations of 100 μM in the culture media. NMU-25-treated
cells showed higher expression of FOXM1 compared to the control cells
(FIG. 15c). Furthermore, FOXM1 was also confirmed to be up-regulated in
LC319 cells expressing exogenous NMU, compared with that of control cells
transfected with mock vector (data not shown).

[0384] We then examined the biological significance of the FOXM1
activation by NMU signaling for growth or survival of lung-cancer cells,
using plasmids designed to express siRNA against FOXM1 (si-FOXM1).
Transfection of si-FOXM1 into A549 or LC319 cells suppressed expression
of the endogenous FOXM1 in comparison to cells containing any of the
three control siRNAs (FIGS. 16a and 16b). In accordance with the reduced
expression of the FOXM1, A549 and LC319 cells showed significant
decreases in cell viability and numbers of colonies (FIG. 16a and b).
These results strongly demonstrated that NMU, by the interaction with
GHSR1b/NTSR1 and subsequent activation of its downstream targets, such as
FOXM1, could significantly affect the growth of lung-cancer cells.

[0385] Microarray data of LC319 cells treated with siRNA for NMU presented
herein proved that NMU signaling pathway could affect the growth
promotion of lung-cancer cells by transactivating a set of downstream
genes involving transcripts whose protein products can function as a
transcription factor and are capable of controlling cell growth or
participating in signal transduction. We provided evidence that the FOXM1
transcription factor is a downstream target of NMU signaling by
additional biological assays. FOXM1 was known to be over-expressed in
several types of human cancers (Teh, M. T. et al., Cancer Res. 62,
4773-4780; van den Boom, J. et al., (2003). Am. J. Pathol. 163,
1033-1043; Kalinichenko, V. V. et al., (2004). Genes. Dev. 18, 830-850).
The "forkhead" gene family, originally identified in Drosophila,
comprises transcription factors with a conserved 100-amino acid
DNA-binding motif, and has been shown to play important roles in
regulating the expression of genes involved in cell growth,
proliferation, differentiation, longevity, and transformation.
Cotransfection assays in the human hepatoma HepG2 cell line demonstrated
that FOXM1 protein stimulated expression of both the cyclin B1 (CCNB1)
and cyclin D1 (CCND1) (Wang, X. et al., (2002). Proc. Nat. Acad. Sci. 99,
16881-16886.), suggesting that these cyclin genes are direct FOXM1
transcription targets and that FOXM1 controls the transcription network
of genes that are essential for cell division and exit from mitosis. It
should be noted that we observed activation of CCNB1 in the majority of a
series of NSCLC we examined and its good concordance of the expression to
FOXM1 (data not shown). On the other hand, it was also demonstrated that
p27 (Kip1) and p19 (Arf) (CDKN2A) interact with FOXM1 and inhibit FOXM1
transcriptional activity (Kalinichenko, V. V. et al., (2004). Genes. Dev.
18, 830-850). The promotion of cell growth in NSCLC cells by NMU might
reflect transactivation of FOXM1, which would affect the function of
those molecular pathways in consequence.

[0386] By immunohistochemical analysis on tissue microarray, we detected
increased expression of NMU protein in the majority of NSCLC (SCC, ADC,
LCC, and BAC) and SCLC samples, but not in normal lung tissues. Since NMU
is a secreted protein and most of the clinical NSCLC samples used for our
analysis were at an early and operable stage, NMU might serve as a
biomarker for diagnosis of early-stage lung cancer, in combination with
fiberscopic transbronchial biopsy (TBB) or blood tests.

[0387] In summary, we have shown that NMU and two newly revealed receptors
for this molecule, GHSR1b and NTSR1, are likely to play an essential role
for an autocrine growth-promoting pathway in NSCLCs by modulating
transcription of down stream target genes. The data reported here
strongly imply the possibility of designing new anti-cancer drugs,
specific for lung cancer, that target the NMU-GHSR1b/NTSR1 pathway. They
also suggest a potential for siRNAs themselves to interfere with this
pathway, as a novel approach to treatment of chemotherapy-resistant,
advanced lung cancers.

INDUSTRIAL APPLICABILITY

[0388] The expression of human genes KIF11, GHSR1b, NTSR1 and FOXM1 are
markedly elevated in non-small cell lung cancer (NSCLC) as compared to
normal lung tissues. Accordingly, these genes can be conveniently used as
diagnostic markers of NSCLC and the proteins encoded thereby may be used
in diagnostic assays of NSCLC.

[0389] The present inventors have also shown that the expression of KIF11,
GHSR1b, NTSR1 or FOXM1 promotes cell growth whereas cell growth is
suppressed by small interfering RNAs corresponding to KIF11, GHSR1b,
NTSR1 or FOXM1 gene. These findings show that each of KIF11, KOC1,
GHSR1b, NTSR1 and FOXM1 proteins stimulate oncogenic activity. Thus, each
of these oncoproteins is a useful target for the development of
anti-cancer pharmaceuticals. For example, agents that block the
expression of KIF11, KOC1, GHSR1b, NTSR1 or FOXM1, or prevent its
activity may find therapeutic utility as anti-cancer agents, particularly
anti-cancer agents for the treatment of NSCLC. Examples of such agents
include antisense oligonucleotides, small interfering RNAs, and ribozymes
against the KIF11, KOC1, GHSR1b, NTSR1 or FOXM1 gene, and antibodies that
recognize KIF11, KOC1, GHSR1b, NTSR1 or FOXM1 polypeptide.

[0390] While the invention has been described in detail and with reference
to specific embodiments thereof, it will be apparent to one skilled in
the art that various changes and modifications can be made therein
without departing from the spirit and scope of the invention.